Drug Delivery: Principles and Applications

DRUG DELIVERY

DRUG DELIVERY: Principles and Applications

BINGHE WANG Georgia State University

TERUNA SIAHAAN University of Kansas

RICHARD SOLTERO DirectPharma, Inc.

A JOHN WILEY & SONS, INC. PUBLICATION

Copyright # 2005 by John Wiley & Sons, Inc. All rights reserved. Published by John Wiley & Sons, Inc., Hoboken, New Jersey. Published simultaneously in Canada. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400, fax 978-646-8600, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services please contact our Customer Care Department within the U.S. at 877-762-2974, outside the U.S. at 317-572-3993 or fax 317-572-4002. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print, however, may not be available in electronic format. Library of Congress Cataloging-in-Publication Data: Wang, Binghe. Drug delivery : principles and applications / Binghe Wang, Teruna Siahaan, Richard soltero. p. cm. Includes index. ISBN 0-471-47489-4 (cloth) 1. Drug delivery systems. 2. Pharmaceutical chemistry. I. Siahaan, Teruna. II. Soltero, Richard. III. Title. RS199.5.W36 2005 2004020585 6150 .6–dc22

Printed in the United States of America

10 9 8 7 6 5 4 3 2 1

CONTENTS

Preface

vii

List of Contributors

ix

1

Factors That Impact the Developability of Drug Candidates: An Overview

1

Chao Han and Binghe Wang

2

Physiological, Biochemical, and Chemical Barriers to Oral Drug Delivery

15

Anna Maria Calcagno and Teruna J. Siahaan

3

Pathways for Drug Delivery to the Central Nervous System

29

Yan Zhang and Donald W. Miller

4

Physicochemical Properties, Formulation, and Drug Delivery

57

Dewey H. Barich, Mark T. Zell, and Eric J. Munson

5

Targeted Bioavailability: A Fresh Look at Pharmacokinetic and Pharmacodynamic Issues in Drug Delivery

73

William F. Elmquist

6

Presystemic and First-Pass Metabolism

83

W. Griffith Humphreys

7

Cell Culture Models for Drug Transport Studies

103

D. Nedra Karunaratne, Peter S. Silverstein, Veena Vasandani, Amber M. Young, Erik Rytting, Bradley Yops, and Kenneth L. Audus v

vi

8

CONTENTS

Prodrug Approaches to Drug Delivery

125

Longqin Hu

9

Receptor-Mediated Drug Delivery

167

Christopher P. Leamon and Philip S. Low

10

Oral Protein and Peptide Drug Delivery

189

Rick Soltero

11

Metabolic Activation and Drug Targeting

201

Xiangming Guan

12

Ultrasound-Mediated Drug Delivery

245

Ka-yun Ng and Terry O. Matsunaga

13

Polycationic Peptides and Proteins in Drug Delivery: Focus on Nonclassical Transport

279

Lisa A. Kueltzo and C. Russell Middaugh

14

Gene Therapy and Gene Delivery

305

Naoki Kobayashi, Makiya Nishikawa, and Yoshinobu Takakura

15

Parenteral Formulation for Peptides, Proteins, and Monoclonal Antibodies Drugs: A Commercial Development Overview

321

John A. Bontempo

16

Pulmonary Drug Delivery: Pharmaceutical Chemistry and Aerosol Technology

341

Anthony J. Hickey

17

Antibody-Directed Drug Delivery

363

Herve´ Le Calvez, John Mountzouris, Kosi Gramatikoff, and Fang Fang

18

Efflux Transporters in Drug Excretion

381

Shuzhong Zhang and Marilyn E. Morris

19

Liposomes as Drug Delivery Vehicles

411

Guijun Wang

20

Regulatory and Intellectual Property Issues in Drug Delivery Research

435

Shihong Nicolaou

Index

443

PREFACE

The pharmaceutical industry, at least the part represented by the major pharmaceutical companies, has undergone a fundamental transformation during the last decade. This transformation has changed the ‘‘silo’’ structure, where different ‘‘steps’’ of the drug discovery process were treated independent of each other, to that of an integrated approach. This transformation was brought about by several factors. One important reason was that about 60% of new chemical entities (NCE) fail clinical trials because of poor pharmacokinetics, undesirable metabolic properties, and toxicity. NCEs do not fail due to a lack of biological activities per se. In addition, the transformation came about because of development in areas such as combinatorial chemistry, high throughput screening, genomics, and proteomics that has allowed for the rapid identification of a large number of biologically active compounds. Therefore, there is a tremendous pressure to have a mechanism to identify a ‘‘winner’’ among a large pool of candidates or ‘‘play out’’ the failures early on. An integrated approach helps to achieve this goal by evaluating various factors such as formulation, permeation, metabolism, and toxicity early in the drug discovery and development process. Such a practice also helps to bring issues that impact development to the awareness of medicinal chemists so that many structural features detrimental to clinical development would be avoided at the designing stage. Among all the factors that effect the clinical development of a NCE, drug delivery occupies a special place. In an integrated drug discovery approach, one has to start considering the delivery properties of a NCE at the design stage. This means that medicinal chemists are the first line of researchers who have to consider this issue. However, most entry-level medicinal chemists are trained as synthetic chemists, and have very little exposure to drug delivery. Without a basic understanding of the issues that effect drug delivery properties, it is hard for the medicinal chemists to address this issue. This book systematically examines various subject areas vii

viii

PREFACE

important to drug delivery, and should be an excellent desk reference for medicinal chemists interested in gaining an overview of this field. In addition, there is a need to strengthen the education of our future medicinal chemists in the area of drug delivery. This book can also serve as a textbook for graduate students or advanced level undergraduate students interested in a career in the pharmaceutical industry. The book starts with chapters that cover general drug delivery issues such as physicochemical and biological barriers, various pathways for drug delivery, formulation, pharmacokinetic and pharmacodynamic issues, metabolism, and cell culture models used in studying drug delivery. Then it moves on to cover specific drug delivery strategies. At the end, we have added one chapter on intellectual property so as to give readers a general idea of how to protect their intellectual property when doing drug delivery research. Each chapter is structured in such a way that it gives an overview of the specific subject, and also goes into details with selected examples so that there is an in depth discussion with extensive references. With this kind of structure, the book will be valuable to both novices and experts. We would like to thank Ms. Neeta Raje for her diligent work in assisting BW in organizing and coordinating the editing and processing of the manuscripts. BINGHE WANG TERUNA SIAHAAN RICHARD SOLTERO

CONTRIBUTORS

Kenneth L. Audus, Department of Pharmaceutical Chemistry, School of Pharmacy, The University of Kansas, 2095 Constant Avenue, Lawrence, KS 66047 Dewey H. Barich, Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, KS 66047 John A. Bontempo, Biopharmaceutical Product Development Consultant, 18 Benjamin Street, Somerset, NJ 08873 Anna Maria Calcagno, Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66047 Herve´ Le Calvez, Abgent Inc., 6310 Nancy Ridge Drive, Suite 106, San Diego, CA 92121 William F. Elmquist, Department of Pharmaceutics, University of Minnesota, Minneapolis, MN 55455 Fang Fang, NexBio, Inc., 6330 Nancy Ridge Drive, Suite 105, San Diego, CA 92121 Kosi Gramatikoff, Abgent Inc., 6310 Nancy Ridge Drive, Suite 106, San Diego, CA 92121 Xiangming Guan, Department of Pharmaceutical Sciences, College of Pharmacy, South Dakota State University, Brookings, SD 57007 Chao Han, GlaxoSmithKline, Collegeville, PA 19426 Anthony J. Hickey, School of Pharmacy, University of North Carolina, Kerr Hall, Chapel Hill, NC 27599 ix

x

CONTRIBUTORS

Longqin Hu, Department of Pharmaceutical Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854 W. Griffith Humphreys, Department of Metabolism and Pharmacokinetics, Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 5400, Princeton, NJ 08543 D. Nedra Karunaratne, Department of Pharmaceutical Chemistry, School of Pharmacy, The University of Kansas, 2095 Constant Avenue, Lawrence, KS 66047 Naoki Kobayashi, Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan Lisa A. Kueltzo, Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, CO 80262 Christopher P. Leamon, Endocyte, Inc., 1205 Kent Avenue, West Lafayette, IN 47906 Philip S. Low, Purdue University, West Lafayette, IN 47907 Terry O. Matsunaga, Imarx Therapeutics Inc., Tucson, AZ 85719 C. Russell Middaugh, Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS 66047 Donald W. Miller, Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198-6025 Marilyn E. Morris, Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, State University of New York, Amherst, NY 14260 John Mountzouris, Abgent Inc., 6310 Nancy Ridge Drive, Suite 106, San Diego, CA 92121 Eric J. Munson, Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, KS 66047 Ka-yun Ng, Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, CO 80262 Shihong Nicolaou, Technology Transfer and Intellectual Property Services, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093 Makiya Nishikawa, Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan Erik Rytting, Department of Pharmaceutical Chemistry, School of Pharmacy, The University of Kansas, 2095 Constant Avenue, Lawrence, KS 66047

CONTRIBUTORS

xi

Teruna J. Siahaan, Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66047 Peter S. Silverstein, Department of Pharmaceutical Chemistry, School of Pharmacy, The University of Kansas, 2095 Constant Avenue, Lawrence, KS 66047 Rick Soltero, Pharma Directions, Inc. Yoshinobu Takakura, Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan Veena Vasandani, Department of Pharmaceutical Chemistry, School of Pharmacy, The University of Kansas, 2095 Constant Avenue, Lawrence, KS 66047 Binghe Wang, Department of Chemistry, Georgia State University, Atlanta, GA 30303 Guijun Wang, Department of Chemistry, University of New Orleans, New Orleans, LA 70148 Amber M. Young, Department of Pharmaceutical Chemistry, School of Pharmacy, The University of Kansas, 2095 Constant Avenue, Lawrence, KS 66047 Bradley Yops, Department of Pharmaceutical Chemistry, School of Pharmacy, The University of Kansas, 2095 Constant Avenue, Lawrence, KS 66047 Mark T. Zell, Pfizer Global Research and Development, Ann Arbor Laboratories, 2800 Plymouth Road, Ann Arbor, MI 48105 Shuzhong Zhang, Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, State University of New York, Amherst, NY 14260 Yan Zhang, Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198-6025

1 FACTORS THAT IMPACT THE DEVELOPABILITY OF DRUG CANDIDATES: AN OVERVIEW CHAO HAN GlaxoSmithKline, Collegeville, PA 19426

BINGHE WANG Department of Chemistry, Georgia State University, Atlanta, GA 30303

1.1. Issues facing the pharmaceutical industry 1.2. Factors that impact developability 1.2.1. Commercial goal 1.2.2. The chemistry efforts 1.2.3. Target validation in animal models 1.2.4. Pharmacokinetics and drug metabolism 1.2.5. Preparation for pharmaceutical products 1.2.6. Remarks on developability criteria 1.3. Drug delivery factors that impact developability References

1.1. ISSUES FACING THE PHARMACEUTICAL INDUSTRY Drug discovery is a long, arduous, and expensive process. It was estimated that the total expenditure for research and development in the U.S. pharmaceutical industries

Drug Delivery: Principles and Applications Edited by Binghe Wang, Teruna Siahaan, and Richard Soltero ISBN 0-471-47489-4 # 2005 John Wiley & Sons, Inc. 1

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FACTORS THAT IMPACT THE DEVELOPABILITY OF DRUG CANDIDATES

was over $20 billion a year in the late 1990s,’ and this figure has been increasing.1 The average cost for every new drug (a new chemical entity, NCE) from research laboratory to patients is a staggering number: $400 to $650 million,2–4 and the whole process may take up to 14 years!5 Because of the high cost, there is tremendous pressure to maximize efficiency and minimize the time it takes to discover and bring a drug to the market. In order to do this, it is necessary to analyze the entire drug discovery and development process and identify steps where changes can be made to increase efficiency and save time. Analyzing the entire drug discovery and development process will help reveal where maximal improvements can be expected with some effort. The entire endeavor of bringing a new drug from idea to market is generally divided into several stages: target/disease identification, hit identification/discovery, hit optimization, lead selection and further optimization, candidate identification, and clinical trials.6 Each stage has many aspects and components. A target is identified early in the discovery period, when there is sufficient evidence to validate the relationship between this target and a disease of interest. Tens of thousands of new compounds are then synthesized and screened against the target to identify a few compounds (hits) with the desired biological activity. Analogs of these selected compounds are then screened further for better activity and optimized in order to identify a small number of compounds for testing in pharmacological models. These efficacious compounds (leads) are further optimized for their biopharmaceutical properties, and the most drug-like compounds (drug candidate, only one or two) are then selected for further development. The drug discovery and development path, with emphasis on the discovery stages, is schematically illustrated in Figure 1.1. Therapeutics areas Unmet needs Ideas, targets

Successful medicine

Molecule

Drug

In vitro activity

Compound library

Candidate selection

Efficacy & developability

Hits

Leads

IND, clinical trial and NDA

Drug candidates

Marketed medicine

Number of compounds 100,000

100

10

1-2

~1

Figure 1.1. A schematic illustration of the drug discovery and development process with the estimated number of compounds shown for each step.

3

ISSUES FACING THE PHARMACEUTICAL INDUSTRY

Of those drug candidates with most drug-like properties, only about 40% make their way to evaluation in humans (Phase I clinical trial).7 Unfortunately, the historical average reveals an almost 90% overall attrition rate in clinical trials;7 in another words, only 1 compound makes it to market from among 10 compounds tested in humans. Results from another statistical analysis gave a similar success rates for NCEs for which an IND (investigational new drug) was filed during 1990–1992.8 This high attrition rate obviously does not produce the long-term success desired by both the pharmaceutical and health care industries. In order to reduce the failure rate, it is necessary to analyze how and where failures occur. More than 10 years ago, Prentis et al.9 analyzed the cause of the high attrition rate based on data from seven UK-based pharmaceutical companies from 1964 to 1985. The results revealed that 39% of the failure was due to poor pharmacokinetic properties in humans; 29% was due to a lack of clinical efficacy; 21% was due to toxicity and adverse effects; and about 6% was caused by commercial limitations. Although not enough detailed information was available, it is believed that some of these causes are interrelated. For instance, toxicity or lack of efficacy can be caused by poor or undesired pharmacokinetic properties. With the understanding that most failure was not due to a lack of ‘‘biological activities’’ per se as defined by in vitro testing, there is a drive to incorporate the evaluation of the other major factors that may potentially precipitate developmental failures in the early drug discovery and candidate selection processes. This is intended to reduce the rate of late-stage failures, which is most costly. This point is further substantiated by the studies indicating that the major cost in drug discovery and development occurs at late stages.10 For example, in a $400 million total R&D cost,4 preclinical research costs probably account for only tens of million dollars, whereas clinical studies cost hundreds of millions of dollars (Figure 1.2). Total R & D Expenses 450 400 US$ million

350 300 250 200 150 100 50 0 1

2

3

4 5 6 Years in R & D

7

8

9

Figure 1.2. Estimated annual expenses based on a hypothetical $400 million total R&D expense for the development of a NCE within a nine-year period (launch in 10th year). Data from Drews, J. and Ryser, S. (1997).4

4

FACTORS THAT IMPACT THE DEVELOPABILITY OF DRUG CANDIDATES

Another factor that is fueling the movement for early integration of multiple disciplines in the drug discovery and development processes is the rapid development of chemical and biological sciences. The past decade has seen tremendous advances in both areas. Advances in combinatorial chemistry, molecular and cellular biology, high-throughput screening, and genomic research have provided both great opportunities and challenges to the pharmaceutical industry. With the rapid development in biological sciences, current interests in therapeutic targets are more focused on rational targets such as receptors, enzymes, and hormones with well-characterized structures and functions. New technologies such as combinatorial chemistry, automation in high-throughput screening, and better instrumentation in bioanalysis have also significantly accelerated the lead identification and discovery process11 for a given target. With these new technologies, large pharmaceutical research organizations are capable of synthesizing and screening several thousand compounds or more in a year or two to find potential drug candidates.12 These efforts typically result in the discovery of many lead compounds or potential candidates for a target in the drug discovery process. Then there is the question of how to pick a winner and how to minimize failures. This requires a thorough evaluation of all the factors that are known to affect the developability of a NEC at the early stages. These factors may include efficacy, pharmacokinetics, pharmacodynamics, toxicology, and drug-drug interactions based on the metabolism and substrate properties of certain transporters and enzymes, as well as physicochemical properties, many of which are related to drug delivery issues. For this reason, a drug discovery and development program is more like a symphony (not just a cross-functional action) of multiple sciences including chemistry, biology, toxicology, clinical science, and pharmaceutical engineering. Under the pressure to reduce the cost and shorten the time needed to bring an NCE to the market, many major pharmaceutical organizations have undergone rapid and drastic changes in the past decade, both in terms of organizational structures and fundamental approaches, in order to develop an integrated approach to drug discovery and development.13 A conference entitled ‘‘Opportunities for Integration of Pharmacokinetics, Pharmacodynamics, and Toxicokinetics in Rational Drug Development’’14 was the landmark event in this fundamental change in the pharmaceutical industry.15 A brand new concept, ‘‘ensuring developability,’’ was introduced and well accepted, which employs criteria for drug development throughout the entire drug discovery and development processes. Under the guidance of such criteria, a drug discovery and development team will not only maximize the chance of success by selecting the best developable drug candidate, but will also play off the failures faster and more cheaply. The paradigm shifts mostly involve the integration of research activities in functional areas such as pharmacokinetics and drug metabolism, pharmaceutical development, safety assessment, and process chemistry into drug discovery and development process in the very early stages of discovery. The inputs from these functional areas, as well as those from clinical, regulatory, commercial, and marketing groups in the early stages, help to minimize costly mistakes in late stages of development and have become more and more important to the success of the

FACTORS THAT IMPACT DEVELOPABILITY

5

drug discovery and development process. Developability is an overall evaluation of the drug-like properties of a NCE. Many of the recent changes in the pharmaceutical industry have been driven by the concept of ensuring developability. These changes, that is, the integration of multifunctional areas in drug discovery and development, ensure that the NCEs of interest will be successful in every step toward the final goal. Below is a brief introduction to the factors that impact developability and a discussion on why the examination of drug delivery issues is very important in helping to ensure the developability of a drug candidate.

1.2. FACTORS THAT IMPACT DEVELOPABILITY In most pharmaceutical companies, many efforts have been made to create a clear framework for selecting compound(s) with minimal ambiguity for further progression. Such a framework is not a simple list of the factors that impact the quality of a drug-like molecule. This framework, which is more often referred as ‘‘developability criteria,’’ is a comprehensive summary of the characteristics, properties, and qualities of the NCE(s) of interest, which normally consist of preferred profiles with a minimally acceptable range. The preferred profile describes the optimal goal for selection and further progression of a candidate, whereas the minimum range gives the acceptable properties for a compound that is not ideal but may succeed. Molecules that do not meet the criteria will not be considered further. Such criteria cover all the functional areas in drug development. Some of the major developability considerations are briefly described in the following subsections. 1.2.1. Commercial Goal It does not need to be emphasized that we are in a business world. Generally speaking, a product needs to be profitable to be viable. Therefore, early inputs from commercial, marketing, and medical outcome professionals are very important for setting up a projective product profile, which profoundly affects the creation of the developability criteria for the intended therapeutics. In general, this portfolio documents the best possible properties of the product and the minimum acceptable ones that may succeed based on the studies of market desires. These studies should be based on the results of professional analyses of the medical care needs, potential market, and existing leading products for the same, similar, or related indications. The following aspects need to be well thought out and fully justified before the commencement of a project: (1) therapeutic strategy; (2) dose form and regimen; and (3) the best possible safety profile, such as the therapeutic window, potential drug interactions, and any other potentially adverse effects. Using the development of an anticancer agent as an example for therapeutic strategy selection, one may consider the choice of developing a chemotherapeutic (directly attacking the cancer cells) versus an antiangiogenic agent (depriving cancer cells of their nutrients), or combined or stand-alone therapy. In deciding the optimal dose form and regimen,

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FACTORS THAT IMPACT THE DEVELOPABILITY OF DRUG CANDIDATES

one may consider whether an oral or intravenous (iv) formulation, or both, should be developed, and whether the drug should be given once daily or in multiple doses. The results of such an analysis form the framework for developing the developability criteria and become the guideline in setting up the criterion for each desired property. For example, pharmacokinetic properties such as the half-life and oral bioavailability of a drug candidate will have a direct impact on developing a drug that is to be administered orally once a day. 1.2.2. The Chemistry Efforts Medicinal chemistry is always the starting point and driver of drug discovery programs. In a large pharmaceutical R&D organization, early discovery of bioactive compounds (hits) can be carried out either by random, high-throughput screening of compound libraries, by rational design, or both. Medicinal chemists will then use the structural information of the pharmacophore thus identified to optimize the structures. Chemical tractability needs to be examined carefully at the very beginning when a new chemical series is identified. Functional modifications around the core structure are carefully analysed. After the examination of a small number of compounds, the initial exploratory structure–activity relationship (SAR) or quantitative SAR (QSAR) should be developed. Blackie et al.16 described how the establishment of exploratory SAR helped the discovery of a potent oral bioavailable phospholipase A2 inhibitor. In this example, numerous substructural changes were made, leading to the most active compounds; this is normally done in parallel with several different chemical series. For medicinal chemists, it is important that many different SARs are considered, developed, and integrated into their efforts at the same time, providing more opportunities to avoid undesirable properties unrelated to their intended biological activities. Such factors, again, may include potential P450 inhibition, permeability, selectivity, stability, solubility, etc. Structural novelty of the compounds (i.e., can this product be patented?), complexity of synthetic routes, scalability (can the syntheses be scaled up in an industrial way?) and the cost of starting materials (cost of goods at the end of the game), and potential environmental and toxicity issues will all need to be closely examined at early stages of the drug discovery and development processes. It is never too early to put these thoughts into action. 1.2.3. Target Validation in Animal Models Although drug discovery efforts almost always start with in vitro testing, it is well recognized that promising results of such testing do not always translate into efficacy. There are numerous reasons for this to happen, some of which are well understood and others that are not. Therefore, target validation in animal models before clinical trials in humans is a critical step. Before a drug candidate is fully assessed for its safety and brought to a clinical test, demonstration of the efficacy of a biologically active compound (e.g., active in an enzyme binding assay) in

FACTORS THAT IMPACT DEVELOPABILITY

7

pharmacological models (in vivo, if available) is considered a milestone in the process of discovering a drug candidate. Many cases exemplify the challenges and importance of pharmacological models. For example, inhibitors of the integrin receptor avb3 have been shown to inhibit endothelial cell growth, which implies their potential as clinically useful antiangiogenic agents for cancer treatment.17 However, the proposed mechanism did not work in animal models, although compounds were found to be very active in vitro.18,19 What has been recognized is that the integrin receptor avb3 may not be the exclusive pathway on which cell growth depends. Its inhibition may induce a compensatory pathway for angiogenesis. Ideally, an in vivo model should comprise all biochemical, cellular, and physiological complexities, as in a real-life system, which may predict the behavior of a potential drug candidate in human much more accurately than an in vitro system. In order to have a biological hypothesis tested in the system with validity, a compound has to be evaluated in many other regards. Knowing the pharmacokinetic parameters such as absorption, distribution, and metabolism in the animal species that is used in the pharmacological model is critical. Showing successful drug delivery in an animal model serves as an important milestone. The pharmacokinetics/pharmacodynamics relationship, systemic and tissue levels of drug exposure, frequency of dosing following which the drug may demonstrate efficacy, and the strength of efficacy are very important factors that may affect further development of an NEC. They are all directly or indirectly related to drug delivery. 1.2.4. Pharmacokinetics and Drug Metabolism Pharmacokinetics and drug metabolism are more often abbreviated as DMPK. The importance of DMPK in drug discovery and development practices is reflected in the statistics of the attrition rate.9 Most of the changes in the pharmaceutical industry during the past decade occurred in DMPK15 and related fields. The overall goal of DMPK in drug discovery and development is to predict the behavior of a drug candidate in humans. Nevertheless, the focus could be different at different stage of the process. Pharmacokinetics (PK) parameters in animal species that will be used in pharmacological (as noted briefly in the previous paragraph) and safety assessment models provide very important insights (systemic and tissue exposures) for those studies. The results of PK studies in several animal species generate the data for physiologically based models or allometric scaling20,21 to predict the basic pharmacokinetic behavior of a compound in humans. Assays using human tissues, cells, and genetically engineered cell lines provide a tremendous amount of information before the real clinical studies begin. Optimizing DMPK developability factors is immensely beneficial for finding the candidate with best potential for success.22 The desirable (or undesirable) biological effects of a drug in vivo normally are directly related to its exposure. One of these factors, namely, the total systemic exposure, maximum concentration, or duration of the concentration above a certain

8

FACTORS THAT IMPACT THE DEVELOPABILITY OF DRUG CANDIDATES

level, is usually used as a parameter that is correlated with the drug’s efficacy and adverse effects.23 The exposure at a given dose is governed by (1) the ability of the body to remove the drug as a xenobiotic and (2) the route by which the drug is delivered. Blood or plasma clearance is often used as a measure of the ability to eliminate a drug molecule from the systemic circulation. A low to moderate clearance molecule is desirable in most situations unless a fast-action, short-duration drug is needed.24 A drug can be directly introduced into the systemic circulation by several methods. However, for convenience and many other reasons, oral dosage forms are preferred in many situations. Therefore, oral bioavailability of the compound is one of the very important developability criteria for oral drug delivery. Many factors affect the oral bioavailability of a drug. These factors will be discussed in detail in several chapters. In addition to clearance and bioavailability, other major pharmacokinetic parameters also should be evaluated. Volume of distribution is a conceptual pharmacokinetic parameter that scales the extent of a drug distributed into the tissues. A well-known parameter, elimination half-life, can be derived from clearance and volume of distribution. It is a very important developability criterion that warrants the desired dose regimen. It should be noted here that half-life must be discussed in the context of a biologically relevant concentration. A purely mathematically derived half-life is sometimes biological irrelevant. Some more definitive explanations and comprehensive discussion of the major pharmacokinetic parameters and their biological relevance have been extensively reviewed.25,26 These parameters should be examined across several different preclinical species to predict the behavior in humans. The DMPK topics will be discussed in Chapters 5 and 6. Inhibition and induction of drug-metabolizing enzymes,27,28 P-glycoprotein (P-gp) substrate property,29,30 plasma protein binding and binding kinetics,31,32 and metabolic stability in the microsomes or hepatocytes from different species including humans,33 as well as the metabolic pathway and the metabolite identified,34 are all very important developability measurements in the assessment of safety, potential drug-drug interaction, and predictability. These factors need to be optimized and carefully examined against developability criteria. Drug metabolism–related issues are outlined and discussed in Chapter 5. The impact of the transporter, including the efflux transporter in drug delivery and the models used to study and address the issues, will be discussed in Chapters 18, 2, and 3. 1.2.5. Preparation for Pharmaceutical Products Before the early 1990s, the solid state, salt form, aqueous solubility, and dosing formulation for agents used in pharmacological, pharmacokinetic, and toxicological studies were not of major concern. However, an inappropriate salt version or solid form may cause potential drug delivery and stability problems (both physicochemically and chemically) during formulation and pharmaceutical engineering. It is now understood that the investigation of the physicochemical properties of an NCE against developability criteria should start early in the R&D processes.

FACTORS THAT IMPACT DEVELOPABILITY

9

Chapter 4 discusses the physicochemical properties that have a major impact on drug delivery. Aqueous solubility is one of the most important physicochemical properties. It is believed that a drug has to be in solution to be absorbed.35 From the pharmaceutical development point of view, the solid state form is another important factor that affects solubility, the dissolution rate, and eventually developability. The solid state form is the determinant of, to some extent, physicochemical stability, intellectual property, and formulation scalability; this factor should be carefully examined and optimized. Change in crystallinerity from different chemical processes, in some cases, results in a big difference in bioavailability when the drug is delivered by a solid dosage formulation. Many of these properties could change when the salt version and form change. The salt with the best solubility, dissolution rate (which therefore could result in the best bioavailability if given as a solid dose), stability, and other properties such as moisture absorption should be selected before a molecule enters full development.36 In situ salt screening is a new technology used to select the right salt form for a drug candidate.37 For instance, the HCl salt38 was formerly almost the default version for a weak base; however, it has been shown in many cases not to be the best.39 Application of these screening processes in early drug development is one of the major steps in integrating pharmaceutical development into drug discovery and development. Preclinical safety assessment (toxicology) is another functional area, which serves as a milestone in drug discovery and development. The NCEs have to be evaluated for their potential genetic toxicity, as well as for acute, short-term, and long-term toxicity. The results are crucial for further development of the compound. Although the principle and importance of toxicology will not be discussed in this book, many efforts in DMPK and pharmaceutics are made to assure drug delivery in the animal models used in toxicological studies. Metabolic profiles of a drug candidate in the species used in the toxicology studies should be compared with those from human tissues for major differences. The profiles are also examined for potential active/toxic metabolite(s). The factors that have an impact on drug delivery will be extensively discussed in the following chapters. Process chemistry is a large functional area that can have major impacts on a drug’s developability, but it will not be covered in this book. Although the developability criteria in this area will not be discussed here, it is important to point out that quite often collaboration with process chemists is also required early on in order to find the right salt and solid state form. 1.2.6. Remarks on Developability Criteria The concept of ensuring developability in drug discovery and development represents an integration of all functional areas that impact the efficiency, success rate, and timetable of a drug’s development. Coordination of these multifunctional, interlinked, parallel, ongoing scientific and technological research activities is a new challenge to the management of a drug discovery and development enterprise.

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FACTORS THAT IMPACT THE DEVELOPABILITY OF DRUG CANDIDATES

Medicinal Chemistry Proc. Chem. Commercial Input Pharmacology Biology/biochemistry Research

Pharmaceutical Development

Safety Assessment

Pharmacokinetics & Drug Metabolism • Therapeutic area • Target

• DMPK developability

• Activity • Selectivity

• Salt form • Solid state

• Scale up • Material Supply

• Efficacy • In vitro safety assessment • Target Validation • In vivo toxicology • Exposure • PK/PD • DMPK developability of Leads • Exposure in Tox species • Drug delivery (salt/solid state)

Figure 1.3. A simplified illustration of the involvement, collaboration, and interrelationship of different functional areas in a preclinical research and development organization. The bullet points summarize the major developability factors examined at different stages.

Figure 1.3 is a simplified scheme of the interrelationship of major functional areas and their roles in drug discovery and development.

1.3. DRUG DELIVERY FACTORS THAT IMPACT DEVELOPABILITY Delivery of a pharmaceutical agent to the systemic circulation, and consequently to the site of action to produce a desired pharmacological effect, is the ultimate goal of drug delivery. The developability of a drug candidate from a drug delivery perspective has become the core of developability criteria in drug development. As discussed in the previous subsections, many other factors in developability criteria are closely related to drug delivery; this holds true from the research laboratory to clinical trails and from early discovery to postmarket development. In order to accomplish this task, one has to overcome numerous barriers that hinder drug delivery. In a biological system, multiple mechanisms exist to protect the system from exposure to almost any foreign substance while preserving nutrient uptake. The physiological arrangement and the chemical and biochemical barriers associated with the physiological structures form the first line of defense. Any drug, delivered by any route, will almost certainly encounter some of these barriers before reaching at the site of action. These barriers, as well as their physiological and biochemical

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functions and their role in drug delivery, will be discussed in detail in Chapter 2. The special situations related to drug delivery to the central nervous system (CNS) is covered in Chapter 3. How a drug molecule interacts with these barriers is very much determined by the properties of the molecule. These properties are the physicochemical and biochemical characteristics of the molecule. In Chapter 4, physicochemical properties and their implications for formulation and drug delivery will be extensively discussed. Pharmacokinetics and pharmacodynamics provide a general approach by allowing mathematical modeling of the interaction of a drug molecule with the entire biological system to predict drug concentrations in the systemic circulation and therefore providing a prediction of pharmacological responses. Better understanding of the system will allow a pharmaceutical scientist to utilize and manipulate the system for the purpose of drug delivery. Chapter 5 discusses the basic principles and topics in pharmacokinetics and pharmacodynamics. Approaches in drug delivery based on an understanding of pharmacokinetic principles are essential in pharmaceutical development. Developability in drug delivery is an overall assessment of all important factors. Take oral drug delivery as an example.40 Solubility is important because a drug molecule has to be dissolved to be absorbed. Some lipophilicity is essential for the molecule to cross cell membranes by diffusion. In order to finally reach the systemic circulation, the molecule has to survive various chemical and biochemical attacks in the gastrointestinal system and the liver. A flow chart describing sequentially the factors that can impact drug delivery is illustrated in Figure 1.4. The order Solubility

Poor Slow

Good

Dissolution rate

• Screen for better salt • Screen for different crystalline • Finer particle size • Further structure modification

Fast

Permeability

Poor

P-gp substrate

No

• Increase lipophilicity

Yes

Good

Stability in stomach

Not stable

Stable

Metabolic stability • Metabolism in intestine • Metabolism in liver

Not stable

• Structure modification • Dose with P-gp inhibitor • Protection by formulation • Screen for more stable compounds

• Metabolite identification • Structure modification

Metabolically stable

GOOD BIOAVAILABILITY Figure 1.4. The evaluation steps of various factors that impact the oral bioavailability of a drug candidate.

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in which these factors are listed could also be the order of logical thinking when one plans to tackle an oral drug delivery problem. It can also be a reference point for other routes of delivery. It is believed that the permeability and metabolic stability of a drug molecule are two major factors in drug delivery or in the prediction of a drug’s absorption41 when the molecule is in solution. Permeability can be further detailed by passive diffusion and transporter-mediated process. Metabolism of a drug molecule in the liver and intestine can be evaluated by in vitro experimental methods. In many cases, in vitro metabolism (intrinsic clearance) can be used to predict in vivo metabolic clearance.42 Drug metabolism–related issues are discussed in depth in Chapter 6. It is obvious that when efflux transporters such as P-gp are involved, the predictability of in vivo clearance using metabolic intrinsic clearance becomes uncertain.43 A more in-depth understanding of drug transporters and their function in combination with our knowledge of drug metabolism will help predict oral absorption.44,45 Transporter-related drug deliver issues, as well as in vivo and in vitro models used to address these issues, are discussed in the following chapters. Although not discussed in detail in this book, in addition to parenteral (e.g., iv infusion) drug delivery, many other routes of drug delivery are developed for convenience, safety, specific targeting, and delivery of special agents. Most of the physiological and biochemical issues discussed in oral and CNS delivery can be extrapolated to the situations in other drug delivery routes. Knowledge of the physiological and biological barriers for each specific delivery route will help medicinal chemists to design drug candidates with optimal drug delivery properties or at least to avoid obvious problems. Prodrug approaches, utilization of metabolic activation to target a specific organ, and taking advantages of a substrate of specific transporters or carriers are some invaluable examples in modern drug delivery. Many of these issues are discussed in various chapters. The aim of this book is to provide a basic understanding of the major issues in drug delivery. More detailed examination of various topics can be found in the references cited. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Price, Waterhouse, Coopers. Pharma 2005. New York, 1998, pp. 1–20. Collins, M. A.; Shaw, I.; Billington, D. D. Drug Des. Discov. 1999, 16, 181–194. Drews, J.; Ryser, S. Nature Biotechnol. 1997, 15, 1318–1319. Drews, J.; Ryser, S. Drug Disc. Today 1997, 2, 365–372. DiMasi, J. A. Clin. Pharmacol. Ther. 2001, 69, 286–296. Kuhlmann, J. Int. J. Clin. Pharmacol. Ther. 1997, 35, 541–552. Venkatesh, S.; Lipper, R. A. J. Pharm. Sci. 1999, 89, 145–154. DiMasi, J. A. Clin. Pharmacol. Ther. 2001, 69, 297–307. Prentis, R. A.; Lis, Y.; Walker, S. R. Br. J. Clin. Pharmacol. 1988, 25, 387–396. Huff, J. L.; Barry, P. A. Emerg. Infect. Dis. 2003, 9, 246–250.

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11. Drews, J. Science 2000, 287, 1960–1964. 12. Hopfinger, A. J.; Duca, J. S. Curr. Opin. Chem. Biol. 2000, 11, 97–103. 13. Railkar, A. S.; Sandhu, H. K.; Spence, E.; Margolis, R.; Tarantino, R.; Bailey, C. A. Pharm. Res. 1996, 13, S-278. 14. Integration of Pharmacokinetics, Pharmacodynamics, and Toxicokinetics in Rational Drug Development. Plenum Press: New York and London, 1993. 15. Lesko, L. J.; Rowland, M.; Peck, C. C.; Blaschke, T. F. Pharm. Res. 2000, 17, 1335–1344. 16. Blackie, J. A.; Bloomer, J. C.; Brown, M. J. B.; Cheng, H. Y.; Elliott, R. L.; Hammond, B.; Hickey, D. M. B.; Ife, R. J.; Leach, C. A.; Lewis, V. A.; Macphee, C. H.; Milliner, K. J.; Moores, K. E.; Pinto, I. L.; Smith, S. A.; Stansfield, I. G.; Stanway, S. J.; Taylor, M. A.; Theobald, C. J.; Whittaker, C. M. Bioorg. Med. Chem. Lett. 2002, 12, 2603–2606. 17. Eliceiri, B. P.; Cheresh, D. A. J. Clin. Invest. 1999, 103, 1227–1230. 18. Miller, W. H.; Alberts, D. P.; Bhatnger, P. K.; et al. J. Med. Chem. 2000, 43, 22–26. 19. Carron, C. P.; Meyer, D. M.; Pegg, J. A.; Engleman, V. W.; Mickols, M. A.; Settle, S. L.; Westlin, W. F.; Ruminski, P. G.; Nickols, G. A. Cancer Res. 1998, 58, 1930–1935. 20. Mahmood, I. Am. J. Ther. 2002, 9, 35–42. 21. Mahmood, I.; Balian, J. D. Clin. Pharmacokinet. 1999, 36, 1–11. 22. Lin, J. H. Ernst Schering Research Foundation Workshop 2002, 37, 33–47. 23. Woodnutt, G. J. Antimicrob. Chemother. 2000, 46, 25–31. 24. Bodor, N.; Buchwald, P. Med. Res. Rev. 2000, 20, 58–101. 25. Benet, L. Z. Eur. J. Respir. Dis. 1984, 65, 45–61. 26. Benet, L. Z.; Zia-Amirhosseini, P. Toxicol. Pathol. 1995, 23, 115–123. 27. Rodrigues, A. D.; Lin, J. L. Curr. Opin. Chem. Biol. 2001, 5, 396–401. 28. Lin, J. H. Curr. Drug Metab. 2000, 1, 305–331. 29. van Asperen, J.; van Tellingen, O.; Beijnen, J. H. Pharm. Res. 1998, 37, 429–435. 30. Wacher, V. J.; Salphati, L.; Benet, L. Z. Adv. Drug Delivery Rev. 2001, 46, 89–102. 31. Tawara, S.; Matsumoto, S.; Kamimura, T.; Goto, S. Antimicrob. Agents Chemother. 1992, 36, 17–24. 32. Talbert, A. M.; Tranter, G. E.; Holmes, E.; Francis, P. L. Anal. Chem. 2002, 74, 446–452. 33. Rodrigues, A. D.; Wong, S. L. Adv. Pharmacol. 2000, 43, 65–101. 34. Baillie, T. A.; Cayen, M. N.; Fouda, H.; Gerson, R. J.; Green, J. D.; Grossman, S. J.; Klunk, L. J.; LeBlanc, B.; Perkins, D. G.; Shipley, L. A. Toxicol. Appl. Pharmacol. 2002, 182, 188–196. 35. Rowland, M.; Tozer, T. N. Clinical Pharmacokinetics: Concepts and Applications. Lippincott Williams & Wilkins: Philadelphia, 1995, chapter 9, pp. 119–136. 36. Morris, K. R.; Fakes, M. G.; Thakur, A. B.; Newman, A. W.; Singh, A. K.; Venit, J. J.; Spagnuolo, C. J.; Serajuddin, A. T. M. Int. J. Pharm. 1994, 105, 209–217. 37. Tong, W.-Q.; Whitesell, G. Pharm. Dev. Technol. 1998, 3, 215–223. 38. Berge, S. M.; Bighley, L. D.; Monkhouse, D. C. J. Pharm. Sci. 1977, 66, 1–19. 39. Gould, P. L. Int. J. Pharm. 1986, 33, 201–217. 40. Martinez, M. N.; Amidon, G. L. J. Clin. Pharmacol. 2002, 42, 620–643. 41. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev. 1997, 23, 3–25.

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42. Houston, J. B. Biochem. Pharmacol. 1994, 47, 1469–1479. 43. Wu, C. Y.; Benet, L. Z.; Hebert, M. F.; Gupta, S. K.; Rowland, M.; Gomez, D. Y.; Wacher, V. J. Clin. Pharmacol. Ther. 1995, 58, 492–497. 44. Benet, L. Z.; Cummins, C. L. Adv. Drug Delivery Rev. 2001, 50, S3–S11. 45. Polli, J. W.; Wring, S. A.; Humpherys, J. E.; Huang, L.; Morgan, J. B.; Webster, L.; Serabjit-singh, C. S. JEPT 2001, 299, 620–628.

2 PHYSIOLOGICAL, BIOCHEMICAL, AND CHEMICAL BARRIERS TO ORAL DRUG DELIVERY ANNA MARIA CALCAGNO AND TERUNA J. SIAHAAN Department of Pharmaceutical Chemistry The University of Kansas Lawrence, KS 66047

2.1. Introduction 2.2. Physiological barriers to drug delivery 2.2.1. Paracellular pathway 2.2.1.1. Tight junctions 2.2.1.2. Adherens junctions 2.2.1.3. Desmosomes 2.2.2. Transcellular pathway 2.3. Biochemical barriers to drug delivery 2.3.1. Metabolizing enzymes 2.3.2. Transporters and efflux pumps 2.4. Chemical barriers to drug delivery 2.4.1. Hydrogen-bonding potential 2.4.2. Other properties 2.5. Drug modifications to enhance transport across biological barriers 2.5.1. Prodrugs and structural modifications 2.5.2. Formulations 2.6. Conclusions References

Drug Delivery: Principles and Applications Edited by Binghe Wang, Teruna Siahaan, and Richard Soltero ISBN 0-471-47489-4 # 2005 John Wiley & Sons, Inc. 15

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2.1. INTRODUCTION The development of various biotechnological drug products (peptidomimetics, peptides, proteins, and oligonucleotides) has brought about new challenges in drug delivery. These challenges are due to the physicochemical properties of the peptides and peptidomimetics and the presence of physiological, biochemical, and chemical barriers. The oral delivery of these molecules has been the focus of many review articles.1–3 This chapter focuses on the various barriers that these types of drugs must overcome to reach their sites of action after oral delivery. The body contains many biological barriers that serve to protect its interior from a variety of external invaders and toxins. The skin is the largest such obstacle, while the blood-brain barrier forms the tightest barrier to penetration of molecules from the bloodstream to the brain. Similarly, for a drug molecule to be orally bioavailable, it has to traverse the epithelial layer of the gastrointestinal tract. Thus, many factors for enhancing the delivery of molecules through this intestinal mucosal barrier must be considered. The various components of the biological barriers will be discussed in greater detail in this chapter. Several different obstacles must be overcome for the delivery of drugs through the intestinal mucosa or the blood-brain barrier. These obstructions to drug delivery can be categorized as physiological, biochemical, and chemical barriers. The physiological barrier in the intestinal mucosa or the blood-brain barrier protects the body from various molecules such as toxins by inhibiting their passage through the barriers. Next, the drug must overcome the biochemical barrier which consists of metabolizing enzymes, that can degrade it. Finally, the drug has to have optimal physiochemical properties for its permeation across the biological barriers. Thus, these various barriers have to be taken into account when designing drugs with improved absorption characteristics.

2.2. PHYSIOLOGICAL BARRIERS TO DRUG DELIVERY The luminal side of the gastrointestinal tract is covered with an aqueous mucus layer that is secreted by the goblet cells. Before reaching the epithelial layer of the intestinal mucosa, a drug molecule must penetrate this mucus layer, which has a thickness that varies from 100 to 150 mm. This mucus layer acts as a filter for molecules with a molecular mass of 600–800 Daltons.3 The mucus is composed of glycoproteins, which trap water within this layer with a turnover rate of 12–24 hours. Drug penetration through this mucus and unstirred water layer is the ratelimiting step before the drug reaches the surface of the enterocytes.3 Immediately below this mucus layer is a single layer of columnar epithelial cells that are joined together by tight intercellular junctions to form a barrier to the systemic delivery of orally administered drugs. This layer of cells is composed of enterocytes, goblet cells, endocrine cells, and paneth cells.4 The number of goblet cells from the small intestine to the distal colon differs; only 10% of the cells in the small intestine are goblet cells, increasing to 24% in the distal colon.5 There are

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four regions of the gastric epithelium from the proximal to the distal stomach— nonglandular stratified squamous, cardiac, glandular proper gastric (fundic), and pyloric. Each region has a different physiological function. The toughness of the stratified squamous region allows it to resist food abrasion, while the cardiac region is responsible for the production of mucus and bicarbonate. Pepsinogen and hydrochloric acid are secreted from the proper gastric region. The pyloric section is associated with the release of gastrin and pepsinogen.6 Both villi and crypts are lined by the epithelial cell layer. The microvilli amplify the surface area of the intestine,7 and absorb nutrients and secrete mucus. The crypts are responsible for cell renewal. The epithelial layer is in immediate contact with the lumen of the gastrointestinal tract. The lamina propria, which functions as a structural support for the epithelial layer, is situated on the basolateral side of the epithelial layer. The lamina propria contains lymph vessels, smooth muscle cells, nerves, and blood vessels, which nourish the epithelium. The muscularis mucosa makes up the deepest layer, which is thought to be involved in contractility.8 A more detailed description of the forces that hold together the epithelial layer is provided below. A drug can cross the intestinal mucosa via several different mechanisms, depending upon its physiochemical properties. Hydrophobic drugs that can partition through cell membranes are more likely to cross the intestinal mucosa through the transcellular pathway (Figure 2.1, Pathway A). Hydrophilic drugs cannot penetrate the cellular membranes; therefore, they must use the paracellular pathway (Figure 2.1, Pathway B). Unfortunately, this pathway is restricted by the presence ˚ can of tight junctions. Only molecules that have a hydrodynamic radius (30% of total liver CYP enzyme)25 and has the broadest range of known substrates.24,26,27 The mechanism of action of the enzymes is a complex multistep process that leads to the biotransformation of substrate, most often to an oxidized product.28–30 The process of oxidation involves high-energy intermediates and often involves the generation of reactive electrophilic intermediates at the enzyme active site that are sometimes released and can react with cellular components.31–35 This process is thought to contribute to the acute or idiosyncratic toxicity displayed by some compounds.32–34 The variability in expression and activity of the CYP enzymes is a major concern in modern clinical pharmacology. This variability is due to a combination of genetic variation, environmental factors, and inhibition or induction by drugs or xenobiotics. Drugs that are cleared via metabolism by a single CYP enzyme often have significant variability in exposure that can have significant consequences for the efficacy and toxicology of the compound. This is especially problematic for drugs that are cleared primarily by CYP3A4 and CYP2D6. CYP3A4 expression is controlled by genetic factors,36 and the enzyme is inhibited and/or induced by an extensive list of pharmaceutical agents, so new drugs that rely on this enzyme for a large percentage of their clearance will likely be the subject of multiple drugdrug interactions.24 CYP2D6 is polymorphically expressed in the human population, and between 1% and approximately 10% of the population are functionally deficient in this enzyme activity and can be defined as poor metabolizers.37 There are also significant populations which overexpress this enzyme.37 Drugs that rely on CYP2D6 for a large portion of their metabolism will likely have widely variable pharmocokinetics due to these factors. Other CYP enzymes either have polymorphic distribution or are subject to induction or inhibition, but the magnitude of the interactions is not as important as those seen with CYP2D6 and CYP3A4. 6.2.2.2. CYP Enzymes in the Intestine Characterization of the CYP content of the human intestine has been challenging, and the exact complement of enzymes present remains unclear.38–40 It is clear that the major drug-metabolizing enzymes present are CYP2C9, CYP2C19, CYP2D6, and CYP3A4. The major enzyme present in intestinal tissue is CYP3A4, and the activity of intestinal microsomes for a variety of substrates has been shown to be in good agreement with activity in liver. The importance of intestinal CYP oxidation to the first-pass metabolism of drugs has been clearly shown with experiments performed during the anhepatic phase of liver transplantation. These experiments have shown that intestinal oxidation is responsible for significant first-pass elimination of cyclosporin and midazolam.41,42 The content of CYP3A4 in the intestine has been shown to be

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quite variable among subjects and contributes to interpatient variability found for CYP3A4 substrates.42 One aspect of intestinal metabolism that must be considered is the relatively small amount of enzyme present relative to liver and the potential for higher drug concentrations during enterocyte transit. This makes the total enzymatic capacity of the intestine lower and more susceptible to saturation than the liver, especially for high-dose drugs.1 Also, the potential for concentration difference may lead to alterations in the enzyme responsible for metabolism in the two organs, as hypothesized for the intestinal and liver metabolism of NE-100.43 There is growing evidence, however, that the P-gp system works in concert with CYP3A4 to effectively limit substrate concentration to the enzyme and minimize the potential for saturation.44–47 One area that has received a considerable amount of recent attention is the inhibition of intestinal CYP enzymes by components of grapefruit juice.48 This effect has been shown to produce substantial increases in the plasma concentrations of a wide variety of CYP3A substrates. 6.2.2.3. CYP Enzymes in the Liver The liver contains the highest quantity of CYP enzymes of any organ and has an impressive capacity to metabolize drugs and xenobiotics. The CYP enzymes of the liver constitute the most important barrier to the entry of drug-like molecules into the systemic circulation. All major CYP2D6 enzymes are present in liver in quantities that are relatively high compared to other organs. The abundance of the major CYP enzymes in the average human liver has been reported to be CYP1A2 (13%), CYP2A6 (4.0%), CYP2B6 (0.2%), CYP2C9/CYP2C19 (18%), CYP2D6 (1.5%), CYP2E1 (6.6%), and CYP3A4/5 (29%).25 6.2.3. Flavin-Containing Monoxygenase Enzymes The flavin-containing monoxygenase enzymes (FMOs) are expressed in the endoplasmic reticulum and are capable of carrying out a variety of oxidative biotransformations. Various aspects of this enzyme family have recently been reviewed.49,50 Typical reactions are the oxidation of nitrogen or sulfur heteroatoms. The major FMO enzyme expressed in humans is FMO3 and is quite abundant in adult liver.51 The enzyme has been shown to metabolize a large number of xenobiotics and drugs in vitro and in vivo, including drugs such as cimitidine, tamoxifen, itopride, and sulindac. There seems to be a high level of interindividual variability in the expression of FMO3, but the exact understanding of the magnitude of the variability is complicated because the enzyme can be degraded during isolation. The variability is thought to be due largely to genetic polymorphisms in the FMO3 gene.51,52 A rare genetic defect in the FMO3 gene has been shown to be the cause of trimethylaminuria.53,54 FMO1 is expressed in intestine and kidney, but the significance of this enzyme for drug metabolism has not been well established.51 The FMO enzymes are not thought to be inducible by small molecules, and no examples of drug-drug interactions due to inhibition of the enzyme have been described.51

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6.3. PHASE II ENZYME SYSTEMS 6.3.1. Glucuronosyl Transferases The uridine 50 -diphosphate glucuronyltransferases (UGTs) are a family of enzymes found in the endoplasmic reticulum that catalyze the transfer of glucuronic acid to nucleophilic sites on drugs and xenobiotics.55,56 The enzymes have broad substrate specificity and will conjugate phenols, carboxylic acids, alcohols, amines, nitrogencontaining heterocycles, and other moieties. UGT enzymes often catalyze the conjugation of metabolites of the CYP enzymes, which leads to their designation as phase II enzymes; however, it is known that the enzymes directly conjugate many substrates. Enzymes of this family play important roles in the metabolic clearance of a number of substrates, including acetaminophen, mycophenolic acid, propofol, morphine, nonsteroidal anti-inflammatories, and fibrates. The major polymorphism in this enzyme family is located in the UGT1A1 gene.57 The condition characterized by a complete lack of functional UGT1A1 activity, either through lack of protein production or production of inactive protein, is called ‘‘Crigler-Najjar syndrome.’’ The more common polymorphism results in a deficiency of UGT1A1 and is known as ‘‘Gilbert’s syndrome.’’ Polymorphisms in UGT enzymes seem to play a role in determining the efficacy and toxicity of the antitumor agent CPT-1158,59 and may be an important determinant of the toxicity of other drugs.60 Polymorphisms have been found for 6 of the 16 functional UGT genes characterized to date, but their functional significance is not as clear as for UGT1A1.57,61 UGTactivity that leads to acylglucuronide formation may be an important pathway for the bioactivation of some compounds to toxic agents.62–64 Although still controversial, this is one major hypothesis for the toxicity seen with a number of nonsteriodal anti-inflammatory drugs. Acylglucuronides present an analytical challenge because of their instability in biological matrices.63 Also, the conjugates are often hydrolyzed in the gut, which can lead to enterohepatic recirculation. 6.3.1.1. Activity in Intestinal Tissue Multiple glucuronosyl transferase enzymes have been detected in human intestinal tissue, but as with the CYP enzymes, only a subset of the liver enzymes are represented. The two enzymes that are clearly expressed are UGT1A1 and 2B7.56 As with CYP enzymes, the most conclusive studies showing the role of intestinal metabolism involve patients in the anhepatic phase of liver transplantation. These patients were shown to produce the glucuronide conjugate of propofol,65 showing that there was extrahepatic metabolism, although the role of the kidney could not be completely ruled out in these experiments. 6.3.1.2. Activity in Liver Tissue The liver contains the highest content of UGT enzymes, and most if not all of the 16 functional UGT genes are expressed.56 The activities of the enzymes expressed in intestine and liver seem to be very similar. 6.3.2. Sulfotransferases Sulfotransferase enzymes (SULTs) catalyze the sulfation of substrates through the transfer of the sulfuryl group of adenosine 30 -phospate 50 -phosphosulfate

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(PAPS).66–69 The substrates for the enzymes are similar to those of the UGTs and include alcohols, phenols, and amine-containing compounds. These enzymes are located in the cytosol and, like other xenobiotic-metabolizing enzymes are made up of a superfamily of genes.68 In humans, approximately 11 SULTs have been identified, with the SULT1 and SULT2 families being the most important for drug metabolism.68 The enzymes metabolize a number of drug substances but also play an important role in the metabolism of endogenous compounds. SULTs are expressed at a high level in the human fetus and may play an important role in detoxifying xenobiotics during early development. Important drug substrates for the SULT enzymes are acetaminophen, minoxidil, and isoproterenol. The SULT enzymes play an important role in the bioactivation of some chemicals through the conjugation of alcohols to form reactive species. This reaction is most notable in the activation of hydroxylamines derived from arylamines.70,71 6.4. OTHER ENZYMES In addition to the enzyme systems mentioned so far, the following enzyme families can also play a significant role in first-pass metabolism: intestinal peptidases,72 alcohol dehydrogenases,73 and N-acetyl transferases.74,75 6.5. TRANSPORTERS Although this review focuses specifically on metabolism, the increasing knowledge of the intimate connections between metabolism and transport must be mentioned. There are many situations in which the interaction of transporters with drug metabolites has been explored and has led to the hypothesis that CYP, and other enzymes and transporters, work in concert to keep xenobiotics from crossing the intestinal epithelium.44–47 Cases in which there seems to be coordinate activity to preferentially export drug metabolites have been found in Caco-2 cells and in vivo with esterase enzymes76,77 and with CYP enzymes.78 Direct excretion of metabolites into the bile via transporter-mediated efflux can be an important determinant of clearance and first-pass extraction.79–81 This phenomenon is probably much more common than it would seem from the literature, at least in part because there are no facile ways to study the effect other than in vivo using bile duct cannulated rats.

6.6. METHODS TO STUDY FIRST-PASS METABOLISM 6.6.1. General There are several in vivo approaches that can be used to study first-pass metabolism in preclinical species, and these, along with in vitro results, can often shed

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mechanistic insight on the problem of incomplete oral bioavailability. Modern liquid chromatography tandem mass spectrometry (LC-MS/MS) measurement of plasma drug concentrations is a tool to rapidly assess oral bioavailability of new candidate compounds and allows for early definition of bioavailability problems. When bioavailability concerns do arise, there are several procedures, discussed below, that can be followed to isolate the factor(s) limiting oral delivery of the compound. 6.6.2. In Vitro Methods to Study First-Pass Metabolism In vitro experiments can sometimes provide valuable insight into what is happening in vivo that is limiting oral bioavailability. The typical experiments, often employed in tandem, to understand bioavailablilty are determinations of compound solubility, membrane permeability, and stability in subcellular fractions. The membrane permeability assays that are most often employed are either a measurement of permeability through an artificial membrane (Parallel artificial membrane permeability assay, PAMPA, is the most common technique) or a cell monolayer (Caco-2, a human colon carcinoma–derived cell line, is the most common cell monolayer). The subcellular fractions most often employed are plasma (for ester-containing compounds) and liver microsomes with the addition of either reduced nicotinamide adenine dinucleotide phosphate (NADPH) or uridine diphosphoglucuronic acid (UDPGA) as cofactor. Hepatocytes are also a very useful in vitro tool that provide a more complete system for studying metabolism. The low availability of high-quality human hepatocytes for study remains a drawback of this approach, although cryopreserved human cells provide an attractive alternative to freshly isolated cells for performing biotransformation studies. All of the assays mentioned can be set up in an automated, medium-throughput system; however, all require a specific assay to measure the compound concentration at the end of the assay, which places significant limitations on throughput. There have been recent attempts to solve the throughput problems inherent in this type of assay by developing generic endpoint assays for metabolic stability, but there has been no clear solution to date. 6.6.3. Prediction of Human Clearance 6.6.3.1. In Vitro–In Vivo Correlations 6.6.3.1.1. Oxidative Biotransformation in Microsomes The rapid determination of pharmacokinetic parameters, solubility, permeability, and in vitro stability in plasma or liver tissue can often provide a reasonable explanation of the mechanisms limiting oral bioavailability. An approach that is often used is to extrapolate the in vitro rate of metabolism to estimate the hepatic clearance using in vitro–in vivo correlation methods.82–86 These methods use in vitro kinetic parameters, usually Vmax =Km or in vitro t1=2 , to determine the intrinsic clearance, which is then scaled to hepatic clearance using the amount of tissue in the in vitro incubation, the weight of the liver, and the well-stirred model for hepatic clearance.

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Care must be used when determining in vitro parameters.87–92 The methods used to extrapolate to the in vivo situation all assume that the drug concentration at the active site of the CYP enzymes will be much less than the Km value. This is a reasonable assumption for most drugs used clinically under typical in vitro experimental conditions; however, experimental design, especially in vitro t1=2 determinations, should take this into account. The incorporation of protein-binding corrections in these calculations has been somewhat controversial. Clearance of ‘‘free drug’’ was included in the first theoretical models for predicting hepatic extraction from in vitro data93 and was the favored method in early in vitro–in vivo correlation attempts. However, the inclusion of protein binding into the scaling equations tends to underpredict actual in vivo values, and generally, better results are achieved when protein binding is left out of the equations.87–89 The reasons for this could be that (1) the intrahepatic concentration of most drugs is closer to the total plasma concentration than the free plasma concentration due to efficient uptake by the liver or (2) protein binding to microsomal proteins may serve to ‘‘cancel out’’ protein binding in plasma, i.e., the free concentration in microsomal incubations is closer to the free concentration in plasma, so the effect of protein binding is already accounted for in the in vitro parameters. Methods used to account for protein binding or to lessen its effect are (1) to determine the free concentration in microsomal incubations and then calculate in vitro parameters based on free drug (this would then be compared to free plasma clearance values) or (2) to use as little protein in microsomal incubations as possible to lessen the effect of binding. The scale-up from in vitro parameters to predict in vivo human clearance of new chemical entities is still difficult prospectively, especially in cases where the CYP kinetic data do not follow classical Michaelis-Menten behavior.94 The uncertainty of this calculation can be somewhat lessened if the same scale-up methods provide accurate results when applied to preclinical animal data. 6.6.3.1.2. In Vitro–in Vivo Correlations: Other Systems This method has most often been applied to scale rates of CYP-mediated oxidative metabolism in microsomal systems but can also be applied to conjugation95,96 or FMO-catalyzed reactions97 or data derived from hepatocytes98 or other in vitro systems. The scaled clearance values can be compared to the values determined in vivo for a single compound or a set of analogs to give some idea of the predictive capacity of the in vitro systems. The use of microsomes along with UDPGA as a cofactor assay to measure UGT enzyme activity has been hampered historically by the fact that this enzymatic activity in microsomes is often in a ‘‘latent’’ form and requires activation by physical or detergent-induced disruption of the membrane matrices. Recently, a generic method involving the addition of the pore-forming peptide alamethicin to overcome the latency exhibited by this enzyme system has been described.99 The inclusion of alamethicin seems to provide a more consistent method of assessing UGT enzyme activity. 6.6.3.2. Allometric Scaling A second method for predicting clearance is through allometric scaling.100–103 The method has been employed for many years to predict

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human pharmacokinetic parameters and has been shown to provide excellent results in cases where the compound is cleared predominantly through renal elimination. The basic method attempts to derive a relationship between animal pharmacokinetic parameters and body weight and then scale that to derive human parameters. As mentioned above, the method is usually employed with renally eliminated drugs because the allometric relationship often breaks down with drugs cleared through metabolism. This is because there is no accounting for interspecies differences in metabolic rates for the compound of interest. Factors such as maximum lifetime potential have been added to the allometric relationship to try to improve the predictions for highly metabolized drugs, but they have met with only limited success. A hybrid method to calculate human clearance using in vitro data as well as animal pharmacokinetic parameters has been described by Lave et al.104 In this method, the authors correct each animal clearance parameter by multiplying the ratio of the in vitro rate of metabolism in that species by the human in vitro rate. The allometric relation is then derived with the corrected animal pharmacokinetic parameters. 6.6.4. In Vivo Methods to Study First-Pass Metabolism The methods outlined above may not lead to a satisfactory determination of the mechanism of incomplete bioavailability, and additional methods may be necessary in some cases to fully characterize the factors responsible. Bile duct cannulated animals provide a powerful model to examine incomplete bioavailability issues, and the rat provides the most flexibility because terminal studies can routinely be done. For compounds that are thought to have dissolution limitations or instability in the gastrointestinal (GI) tract, the GI tract can be removed at the end of the experiment and the contents assayed for drug and metabolites. The amount of parent in bile and urine can be quantitated by LC-MS/MS, and this method can also be used for any metabolites when authentic standards have been prepared. Alternatively, bile and urine metabolites can be estimated with ultraviolet highperformance liquid chromatography (HPLC-UV) using the extinction coefficient of the parent. These measurements will begin to define the total absorption of the compound and how it relates to the systemic bioavailabilty. The availability of radiolabeled compound at this point makes the bile duct cannulated rat experiment especially powerful. An interesting new methodology for getting early estimates on total drug-related material is through the use of quantitative 19Fnuclear magnetic resonance (19F-NMR).105 In this technique, the 19F-NMR signals in the chemical shift region of the parent signals are recorded and integrated for intact samples of urine and bile. The integrated signals are compared with standards and allow for quantitation of drug-related material. The technique has been shown to produce results that compare favorably to those achieved with standard scintillation counting techniques and, with the prevalence of fluorine-containing compounds in modern drug discovery, could become a useful technique for discovery work. A second in vivo model system that is very useful in dealing with the problem of low oral bioavailability is portal vein cannulated animals. There are two ways

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this experiment can be conducted to determine hepatic extraction: (1) measure the systemic plasma concentration after oral, portal vein, and systemic administration or (2) measure portal vein and hepatic vein concentrations after an oral dose. Both methods yield information on hepatic extraction and the percentage of dose reaching the portal circulation (the product of the fraction absorbed and the fraction metabolized by the gut wall). Both the bile duct cannulated model and the portal vein cannulated model can be combined with a number of methods for modulating absorption or metabolism to ask specific mechanistic questions regarding stability, permeability, and metabolism. The following are several methods that can be used to modulate metabolism in vivo. The most commonly used method to modulate oxidative metabolism in vivo is to coadminister either ketoconazole or 1-aminobenzotriazole106 to inhibit CYP enzymes. Both of these compounds will inhibit intestinal and liver CYP enzymes after oral administration. Care must be exercised when using ketoconazole for this purpose because the compound also has effects on transporters, and these effects may make interpretation of results ambiguous. Alternatively, a CYP inducer can be coadministered to determine the effect on the clearance of the compound of interest. This can be accomplished with one of many known inducers of CYP enzymes. For inhibition of esterase enzyme activity, bis-[p-nitrophenyl] phosphate (BNPP) can be coadministered because this compound does not inhibit cholinesterase activity.107 Intestinal peptidases can be inhibited by a variety of specific inhibitors108 such as bestatin for aminopeptidase109 or aprotinin for chymotrypsin110 in in vitro or in vivo experiments, but the studies must be done by examining one peptidase at a time or by using inhibitor ‘‘cocktails.’’ Methods for inhibiting phase II enzymes in vivo are not as well defined, since in most cases good inhibitors of the enzymes have not been identified. It is possible to deplete co-factor stores by co-administering large amounts of substrate to ask mechanistic questions, although this method is most easily applied to SULTenzymes.111 Many phase II enzymes are subject to induction, which may be an avenue available for modulating enzyme activities in vivo. 6.6.5. Screening Strategies The approach often taken in candidate optimization is to try to isolate bioavailability problems for a member of a chemotype of interest with a combination of in vitro and in vivo experiments and then to try to devise rapid techniques to screen for the liability (Figure 6.1). This approach relies on a good deal of up-front work to fully understand the bioavailability limitations and periodic checking of the property to ensure that the screen is providing reliable results (Figure 6.1). Systematic decision trees can be employed to allow clear pathways for evaluation of new compounds (Figure 6.2). Alternatively, screens can be run in parallel so that all information is generated for all compounds (Figure 6.2). This strategy makes workflows simpler and increases efficiency but can lead to information overload and complex decision making. For metabolic stability screens to be most effective, the screens must be tightly linked to some means of gathering information on metabolite structure. Methods

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Figure 6.1. Scheme for incorporation of ADME-based ‘‘developability’’ screens during the candidate optimization phase of the drug discovery process.

for rapidly determining metabolite molecular weight and limited structural information have improved dramatically and allow this approach to be routinely employed.112 The purpose of this approach is to allow the identification of metabolic ‘‘soft spots,’’ which can then be altered to produce compounds with improved metabolic stability. The literature on successful structural modification to increase stability has recently been reviewed.7 6.6.6. In Silico Methods to Study First-Pass Metabolism Metabolism prediction through in silico methods may be possible in the future, although a great deal of technical development is needed before it is a viable alternative.113 Predictive metabolism programs may allow metabolism scientists and medicinal chemists to obtain metabolic rate or site of metabolism information prior to the first synthesis of compounds. The majority of the methods in this area that have been published to date fall into four categories: (1) expert systems designed to identify probable sites of metabolism based on comparison to database information,114,115 (2) programs to predict the site of metabolism based on molecular orbital theory,116 (3) programs to predict the rate of metabolism based on physical chemical attributes of the molecule and quantitative structure–activity relationship (QSAR) development,117–123 and (4) prediction of the site of metabolism based on knowledge of the CYP active site.124,125

95

Receptor binding

Receptor binding

ADME screening panel

Receptor binding

IC50< 50 nM?

IC50< 50 nM?

yes

yes

yes

yes

Metabolic stability

ADME screening panel

Acceptable ADME properties?

IC50< 50 nM?

In vivo efficacy

Stability sufficient?

yes

yes

Acceptable ADME properties?

In vivo efficacy

In vivo efficacy

Acceptable ADME properties?

ADME screening panel

yes

Figure 6.2. Strategies for the design of decision trees for new compound evaluation. ADME-based screens can be incorporated in many places in a decision tree, three examples are illustrated: A, ADME screens are run as a panel in parallel with early activity screens, compounds proceed to further biological screens after meeting set criteria; B, ADME screens are run after a compound meets set potency criteria and compounds proceed to further biological screens after meeting set ADME criteria; C, critical ADME screen (metabolic stability in this case) is run on compounds that meet potency criteria and then full ADME screen panel is run before compounds proceed.

New compounds

(c)

New compounds

(b)

New compounds

(a)

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Attempts to calculate the site of metabolism based on molecular orbital theory have met with some success. Currently the state-of-the-art method is to calculate an electron density map of molecules using quantum mechanics and then calculate a steric factor using knowledge of the CYP active site.116 Taken together these two parameters have successfully predicted the sites of oxidation for limited sets of molecules.116 This approach may be dramatically enhanced as the crystal structures of more mammalian CYP enzymes become available.126–128 The QSAR approach revolves around the calculation of molecular descriptors as a means of calculating the rate at which a molecule will be metabolized.117–123 This QSAR approach to help define the drug-like properties of a molecule could have a dramatic impact on early drug discovery and allow refinement of early chemical synthesis and library design. 6.7. RATIONALE FOR OPTIMIZATION OF CLEARANCE PROPERTIES OF NEW CHEMICAL ENTITIES The major reason to optimize the metabolism of a new chemical entity is that the clearance properties can be matched to the indication. For most indications involving chronic oral therapy, that means minimizing clearance through minimization of metabolism (this is not true in all cases; some indications may require a short duration of action and thus rapid metabolism). Other benefits of understanding and reducing metabolism are: 1. decreased clearance, which may translate into a lower overall dose; 2. lower rates of formation and overall amounts of reactive intermediates, which may mediate acute or idiosyncratic toxicities; 3. an increased pharmacokinetic half-life, which will hopefully translate into a longer duration of action, less frequent dosing, and better patient compliance; 4. better understanding of the extrapolation of animal data to humans, making human dose projections more reliable and reducing the risk upon entry into clinical development (this is especially important in cases where prodrugs are being developed); 5. lower risk of drug-drug interactions; even if the compound is still dependent on metabolism for the bulk of its clearance, low-clearance drugs are less susceptible in drug-drug interaction caused by the coadministration of inhibitor; 6. lower risk of drug-food interactions due to the reduced dose; 7. decreased formation of metabolites that may have pharmacological activity against the target or may have significant off-target activity. 6.8. SUMMARY The past several decades have witnessed an explosion in our knowledge of drugmetabolizing enzymes. It is now possible to fully characterize and predict the

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metabolic fate of new chemical entities in humans with reasonable certainty. The in vitro methods used to do this, i.e., human liver microsomes, expressed enzymes, and cryopreserved or freshly isolated hepatocytes, can be adapted to be run in medium-throughput fashion and allow the metabolic properties of new compounds to be optimized during the discovery phase. Also, medium-throughput screens for bioavailability are now possible with rapid quantitation by LC-MS/MS techniques. These techniques must continue to evolve to allow metabolism scientists to keep pace with the increasing speed of drug discovery. The increasing complexity of the process will place ever greater dependence on information management, and the ability to turn that information into knowledge will be critical. This is especially true when one considers the advances in other fields of ADME-related research, such as transporters, interactions with nuclear hormone receptors, and so on, that need to be characterized during the discovery of a drug. This complexity ensures that the field of drug metabolism will continue to play an increasing role in the endeavor to provide high-quality drug candidates that will become safe, highly efficacious medicines.

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105. Lenz, E. M.; Wilson, I. D.; Wright, B.; Partridge, E. A.; Rodgers, C. T.; Haycock, P. R.; Lindon, J. C.; Nicholson, J. K. J. Pharm. Biomed. Anal. 2002, 28, 31–43. 106. Balani, S. K.; Zhu, T.; Yang, T. J.; Liu, Z.; He, B.; Lee, F. W. Drug Metab. Dispos. 2002, 30, 1059–1062. 107. Buch, H.; Buzello, W.; Heymann, E.; Krisch K. Biochem. Pharmacol. 1969, 18, 801–811. 108. Aungst, B. J. J. Pharm. Sci. 1993, 82, 979–987. 109. Agu, R. U.; Vu Dang, H.; Jorissen, M.; Willems, T.; Kinget, R.; Verbeke, N. Int. J. Pharm. 2002, 237, 179–191. 110. Tozaki, H.; Emi, Y.; Horisaka, E.; Fujita, T.; Yamamoto, A.; Muranishi, S. J. Pharm. Pharmacol. 1997, 49, 164–168. 111. Kim, H. J.; Cho, J. H.; Klaassen, C. D. J. Pharmacol. Exp. Ther. 1995, 275, 654–658. 112. Watt, A. P.; Mortishire-Smith, R. J.; Gerhard, U.; Thomas, S. R. Curr. Opin. Drug Discov. Dev. 2003, 6, 57–65. 113. van de Waterbeemd, H.; Gifford, E. Nat. Rev. Drug Discov. 2003, 2, 192–1204. 114. Erhardt, P. W. Drug Metabolism: Databases and High-Throughput Testing During Drug Design and Development. IUPAC and Blackwell Science: Boston, 1999. 115. Hawkins, D. R. Drug Discov. Today 1999, 4, 466–471. 116. Jones, J. P.; Mysinger, M.; Korzekwa, K. R. Drug Metab. Dispos. 2002, 30, 7–12. 117. Andrews, C. W.; Bennett, L.; Yu, L. X. Pharm. Res. 2000, 17, 639–644. 118. Ekins, S.; Obach, R. S. J. Pharmacol. Exp. Ther. 2000, 295, 463–473. 119. Yoshida, F.; Topliss, J. G. J. Med. Chem. 2000, 43, 2575–2585. 120. Ekins, S.; de Groot, M. J.; Jones, J. P. Drug Metab. Dispos. 2001, 29, 936–944. 121. Lewis, D. F.; Modi, S.; Dickins, M. Drug Metab. Rev. 2002, 34, 69–82. 122. Long, A.; Walker, J. D. Environ. Toxicol. Chem. 2003, 22, 1894–1899. 123. Taskinen, J.; Ethell, B. T.; Pihlavisto, P.; Hood, A. M.; Burchell, B.; Coughtrie, M. W. Drug Metab. Dispos. 2003, 31, 1187–1197. 124. Lewis, D. F. Drug Metab. Rev. 2002, 34, 55–67. 125. Lewis, D. F. Pharmacogenomics 2003, 4, 387–395. 126. Williams, P. A.; Cosme, J.; Sridhar, V.; Johnson, E. F., McRee, D. E. J. Inorg. Biochem. 2000, 81, 183–190. 127. Williams, P. A.; Cosme, J.; Ward, A.; Angove, H. C.; Vinkovic, D. M.; Jhoti, H. Nature 2003, 424, 464–468. 128. Tennant, M.; McRee, D. E. Curr. Opin. Drug Discov. Dev. 2001, 4, 671–677.

7 CELL CULTURE MODELS FOR DRUG TRANSPORT STUDIES D. NEDRA KARUNARATNE, PETER S. SILVERSTEIN, VEENA VASANDANI, AMBER M. YOUNG, ERIK RYTTING, BRADLEY YOPS, AND KENNETH L. AUDUS Department of Pharmaceutical Chemistry, School of Pharmacy The University of Kansas, 2095 Constant Avenue, Lawrence, KS 66047

7.1. Introduction 7.2. General considerations 7.3. Intestinal epithelium 7.3.1. The intestinal epithelial barrier 7.3.2. Intestinal epithelial cell culture models 7.4. The blood-brain barrier (BBB) 7.4.1. The blood-brain endothelial barrier 7.4.2. BBB cell culture models 7.5. Nasal and pulmonary epithelium 7.5.1. The respiratory airway epithelial barrier 7.5.2. The nasal epithelial barrier and cell culture models 7.5.3. The airway epithelial barrier and cell culture models 7.5.4. The alveolar epithelial barrier and cell culture models 7.6. The ocular epithelial and endothelial barriers 7.6.1. The corneal epithelial barrier and cell culture models 7.6.2. The blood-retinal epithelial barrier and cell culture models 7.7. The placental barrier 7.7.1. The syncytiotrophoblast barrier 7.7.2. The syncytiotrophoblast barrier and cell culture models

Drug Delivery: Principles and Applications Edited by Binghe Wang, Teruna Siahaan, and Richard Soltero ISBN 0-471-47489-4 # 2005 John Wiley & Sons, Inc. 103

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7.8. The renal epithelium 7.8.1. The renal epithelial barrier 7.8.2. Renal epithelial cell culture models 7.9. Conclusions References

7.1. INTRODUCTION In pharmaceutical research there is a need for cell-based assays that have some predictive capability with regard to tissue permeability properties of lead compounds. These assays are generally used after high-throughput screening procedures have identified compounds with the desired pharmacological properties. Cell-based assays can be used to identify those leads that have the desired properties with regard to tissue permeability, and then animal models may be used as a final screen. This approach is not only more cost-effective but also minimizes the use of animal resources. The purpose of this chapter is to present overviews of a selection of the major endothelial and epithelial barriers to drug delivery for which there are either primary culture or cell line systems that recapitulate the characteristics of the in vivo barrier. Our objective is to define some general characteristics of cell culture models and highlight the more commonly applied primary cell cultures and cell lines in use today. Specifically, we focus on cell culture models for the intestinal epithelium, bloodbrain barrier, pulmonary and nasal epithelium, ocular epithelium, placental barrier, and renal epithelium. Renal epithelium was included here primarily because some cell lines derived from this tissue [e.g., Madin-Darby canine kidney cells (MDCK)] are often used as surrogates for other barriers by pharmaceutical scientists. We have arbitrarily chosen to exclude the skin and liver from the scope of this overview. However, it should be noted that hepatocyte cell culture models, for example, are becoming more widely available and have been the subject of recent reviews.1,2

7.2. GENERAL CONSIDERATIONS Technologies for isolating and cultivating endothelial and epithelial cells from many of the tissue interfaces influencing drug distribution have rapidly evolved in the past 20 years. As a consequence, the pharmaceutical scientist now has an assortment of cell culture systems to characterize fundamental drug delivery processes at the biochemical and molecular levels in major epithelial and endothelial barriers. Cell culture models are described as either primary cultures or cell lines. Primary cultures are generated by the growth of cells that migrate out from a fragment of tissue or by growth of cells isolated by either enzymatic or mechanical dispersal of tissue.3 A primary culture may be subcultured or passaged by harvesting the cells

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Dissection of specific organs or tissues

Fine dissection and/or chopping

Enzyme digestion to separate and disperse cells

Primary cell cultures

Primary explant tissue cultures

Collection, enzyme digestion to separate and disperse cells Passage #1 cell cultures

Figure 7.1. General methods for establishing primary explant tissue, primary cell, and passaged cell culture models from animal tissues.

and redistributing (i.e., splitting) them on new growth surfaces. Once a primary culture is passaged, it is known as a ‘‘cell line’’ (Figure 7.1). Generally, subsequent passaging of the cells selects for cells with rapid growth, and by the third passage, the cultures are more stable. Most cell lines may be stable for a limited number of passages. A continuous cell line that may be grown for essentially unlimited passages may occur through in vitro transformation and may arise spontaneously or through chemical or viral induction. Cell cultures derived from most normal tissues rarely give rise to continuous cell lines spontaneously.3 Researchers prefer to work with continuous cell lines because of the convenience and probability of working with a homogeneous cell population versus primary cultures that have to be continuously generated and can vary significantly due to inconsistencies among different preparations. The appropriateness of any cell culture model used to study drug delivery processes, whether a primary or a cell line, should be based on certain basic criteria. These include but are not limited to the presence of a restrictive paracellular pathway that allows effective characterization of transcellular permeability, the presence of physiologically realistic cell architecture reflective of the tissue barrier of interest, the expression of functional transporter mechanisms representative of the tissue barrier of interest, and the ease, convenience, and reproducibility of the culture methods.4 However, pharmaceutical scientists must realize that cell culture models in use today are not ideal with respect to these criteria, and there will be advantages and limitations with any choice.

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1

Plastic growth surface in multiwell plates

2

3

Permeable support (e.g., polycarbonate membrane with 0.4 µm pores)

Donor 0.5 ml Receiver 1.5 ml

Cell suspension

Donor 3.0 ml

Thermostated water jacket

Receiver 3.0 ml

Physiological medium Stir bar

Figure 7.2. General uptake and transport configurations for cell culture models. Depending on the cell type, cell suspensions may be (1) seeded into and grown on plastic surfaces for uptake studies, (2) seeded and grown on permeable supports in TranswellTM-type systems, or (3) seeded and grown on permeable supports that can be placed in a side-by-sideTM diffusion apparatus.

Part of the selection of any cell culture model will also include consideration of an appropriate transport configuration (Figure 7.2) and the quantitative processes necessary to appropriately analyze transport data.5 Options generally available to the pharmaceutical scientist include growth of cells on multiwell plastic dishes for assessment of cellular uptake; this provides for analysis of only one-half of the transcellular transport process. This format is favored for high-throughput types of assays where quick assessment of cellular uptake, retention, or efflux of materials can be quickly assayed with the appropriate markers. More likely, one will consider either the Transwell or side-by-side types of configurations, which provide the advantage of allowing evaluation of transcellular transport and access to both apical and basolateral surfaces of cells grown on permeable supports. Criteria for selection of an appropriate transport model include the solute’s properties, sufficient stirring conditions, filter (permeable support) properties, assay conditions, matrix (growth surface treatments), assay medium properties, and the cell type chosen. Further elaboration on each of these points and the quantitative evaluation of

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transport processes are beyond the scope of this short review. Readers are directed to the extensive review by Ho et al.5 for further details. 7.3. INTESTINAL EPITHELIUM 7.3.1. The Intestinal Epithelial Barrier The general desire for orally administered drugs that are convenient, affordable, and highly bioavailable dictates that the basic characteristics of the intestinal barrier are fully elucidated. Many promising drug candidates are developed at great expense, only to be removed from consideration due to poor oral bioavailability; this is due, in large part, to poor intestinal absorption. Implementation of an accurate model of this barrier at the earliest possible stage of drug design and development will provide valuable information and result in significant economic advantage. A monolayer of multiple cell types comprises the intestinal epithelium. Enterocytes, the absorptive cells, are the main focus in drug delivery issues. In addition to enterocytes, there are undifferentiated crypt cells, mucus-secreting goblet cells, and M cells, which sample the gut lumen for the lymphatic system.6 However, it is the columnar villar enterocyte that forms the primary barrier to absorption. An extensive network of microvilli on the apical surface of villar enterocytes provides a vast amount of surface area for absorption of nutrients, pharmaceuticals, and xenobiotics.6,7 Drugs can cross the intestinal epithelial barrier in a number of ways. They may permeate either through the cell (transcellular) or between adjacent cells (paracellular). Enterocytes have tight intercellular junctions that restrict paracellular transport to small hydrophilic molecules.7 These cells possess active and facilitative transporters for nutrients, as well as an array of efflux transporters [e.g., P-glycoprotein (P-gp) and related transporters] and enzymes (e.g., cytochrome P450 type 3A4) that restrict transcellular absorption. Transcytotic transport of macromolecules is possible, but compounds are often destroyed in lysosomes. With the exception of M-cells, transcytosis is not considered a major mechanism of the transcellular pathway for absorption of macromolecules across gastrointestinal epithelium.6 7.3.2. Intestinal Epithelial Cell Culture Models Many in vivo and in vitro techniques exist to model the intestinal barrier, but recent trends are toward an inexpensive in vitro model that accurately represents human oral absorption of pharmaceuticals.6,8 Multiple cell culture models have been developed, each with its own benefits and drawbacks. In order to effectively assess drug transport, a cell line must form polarized monolayers of differentiated enterocytes. In addition, the cells must retain the morphological and biochemical characteristics of the human intestine.8,9 Primary cultures of human enterocytes have low viability and do not differentiate or form monolayers.10 Immortalized cell lines have been developed with mixed results.11 The Caco-2 cell line, derived from a human colon carcinoma, is the cell culture

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model most extensively utilized for studies of oral bioavailability. Isolated in 1974, it was not employed as a model of oral drug delivery until the late 1980s.12 Caco-2 cells are easily grown in tissue culture, and they form differentiated, polarized monolayers on polycarbonate membranes. The monolayer formed differs from the human intestinal barrier in several ways. Since goblet cells are absent from culture, there is no mucus layer on the apical surface. Intestinal mucosa is considered an additional barrier to drug delivery, so Caco-2 permeability studies may yield artificially high values for certain compounds.9 Biochemical and morphological characteristics are similar to those of enterocytes but are not identical.11–17 Caco-2 cells must be grown for 20 days to form a fully polarized monolayer suitable for drug transport studies. Compared to other cells lines such as MDCK,18,19 which can be established and used for studies in less than a week, Caco-2 cells are labor-intensive and expensive to maintain. For that reason, MDCK cells have begun to be used as a surrogate for Caco-2 cells in screening compounds that have enhanced permeation by simple passive diffusion. There have been attempts to reduce the time necessary to form competent Caco-2 monolayers to 3–5 days, but these efforts have met with limited success.20 At this time, Caco-2 cells are still the most convenient model that provides reasonable in vivo–in vitro correlations. Caco-2 is particularly helpful in predicting the apparent permeabilities of passively absorbed drugs. Carrier-mediated processes will differ among cell lines due to differences in expression levels of transporters. Even if permeability values differ from the actual permeability in the intestine, Caco-2 is a valuable tool for predicting the rank order of permeability for a class of compounds. The differences in tight junctions between Caco-2 and enterocytes in vivo make prediction of paracellular permeability difficult.1 Caco-2 also expresses efflux transporters, such as P-glycoprotein, and CYP450 enzymes that limit oral bioavailability and cause potential drug-drug interactions. Such interactions are not predictable based on current models of transportersubstrate interactions, so Caco-2 cells represent a valuable tool with which to obtain this information.20 Caco-2 cells are currently used at all levels of pharmaceutical research and development. Automation technologies allow tissue culture labs to easily maintain a large number of Caco-2 cells as well as to perform numerous permeability studies without the introduction of many common human errors. Combinatorial chemistry provides vast arrays of compounds, and Caco-2 assays can be used to assess potential permeability and metabolic issues before much money is invested in the candidate.6 These cells are also helpful at the formulation stages. Very little is known about the effect of excipients on intestinal permeability; therefore, it is advantageous to measure the permeation of the active ingredients in the presence of excipients,20 as revealed in recent studies.21 It is apparent that no single method is sufficient for studying or predicting drug absorption across the intestinal epithelium.6 Consequently, simple algorithms based on physicochemical values are only marginally successful in predicting permeability, but the use of Caco-2 data increases predictive capabilities.20 Thus, Caco-2

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permeability values are also helpful in developing in silico models and, in combination, are more useful in predicting drug absorption.6

7.4. THE BLOOD-BRAIN BARRIER (BBB) 7.4.1. The Blood-Brain Endothelial Barrier The BBB controls the exchange of nutrients, xenobiotics, and drugs between the brain and the systemic circulation. The BBB is a significant obstacle to achieving optimal concentrations of desirable therapeutics in the central nervous system, including agents for the treatment of stroke, cancer, Alzheimer’s disease, and human immunodeficiency virus-1 (HIV-1) infection.22 The BBB is localized to the cerebral microvascular endothelial cells and is composed of at least four major components. The first component is the endothelial cells, which differ from their counterparts in peripheral organs in that those in the brain have few pinocytic vesicles and exhibit limited transcytotic activity. Tight junctions effectively seal the spaces between the endothelial cells and restrict paracellular movement of molecules. Thus, the tight junctions form another component of the BBB.23 These intercellular junctions are considered to be dynamic, and permeability can be regulated by several mechanisms.24 Another component of the BBB, and probably the least understood, is the metabolic activity present in the endothelial cell. The presence in endothelial cells of some of the members of the cytochrome P450 family of drug-metabolizing enzymes could restrict access of pharmaceutical agents to the central nervous system (CNS).25 Numerous transmembrane transporters in the endothelial cells are part of the fourth component of the BBB, and are probably the component of greatest interest to researchers in the pharmaceutical sciences. Generally facilitative in nature, transporters at the BBB are involved in the uptake of substances (such as glucose, biotin, amino acids, and various other nutrients and xenobiotics) from the extracellular environment.26 Their importance is emphasized by studies targeting a select group of these transporters to deliver agents that would not normally penetrate the BBB to the CNS; this involves a strategy of conjugation of the agents to natural substrates.22 Other transporters are involved in the active efflux of substances from the endothelial cells. The best-characterized efflux transporter present in the BBB is P-gp, an efflux transporter that was first identified because of its association with multidrug-resistant cancer.27 P-gp is located on the circulation (blood) side of the endothelial cell. It has a very broad substrate specificity and has been shown to be responsible for the exclusion of many compounds from the brain, including HIV-1 protease inhibitors, vincristine, vinblastine, and taxol.27,28 Members of the multidrug resistance–associated protein (MRP) family of transporters are also present in brain microvascular endothelial cells. The mRNAs from MRP 1, 4, 5, and 6 have been identified in bovine brain microvascular endothelial cells (BBMECs), but their functional activities in this tissue remain to be confirmed.29

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7.4.2. BBB Cell Culture Models Currently, there are two primary cell cultures that are used in both academic and industrial settings: BBMECs and porcine brain microvascular endothelial cells (PBMECs).4 Over the past two decades, BBMECs have become a very widely used in vitro cell culture model of the BBB. These cells form monolayers with tight junctions and retain expression of many transporters and enzymes typical of the BBB.4 Large quantities of the cells can be isolated from beef brains using material obtained from freshly sacrificed animals. Brain microvessel-enriched gray matter serves as a source of cells that are isolated by enzymatic digestion and purified by centrifugation. The cells can be stored at
80 C for several weeks; for longer periods they should be stored in liquid nitrogen. For uptake studies the cells can be seeded on tissue culture plates, whereas for transport studies the cells can be seeded on polycarbonate membranes. The cells isolated using this procedure form monolayers with relatively tight junctions.4,30 Detailed protocols for these procedures have been provided by Audus et al.30 In primary culture, BBMECs are not exposed to the influence of other cells that may impact their development. In this regard, glial cells and astrocytes are thought to be of particular importance.24,31 Two basic strategies have evolved to try to improve the faithfulness of BBMECs in primary culture to the BBB: growth of BBMECs in astrocyte-conditioned media and growth of BBMECs in proximity to astrocytes in such a way that soluble factors secreted by the astrocytes can influence the BBMECs. One way in which this can be accomplished is by growth of the BBMECs in a Transwell, and growth of the astrocytes in the bottom of the well into which the Transwell is placed. Most of the changes reported by those using astrocyte coculture, or astrocyte-conditioned media, relate to a decrease in paracellular permeability, as demonstrated by increases in transepithelial electrical resistance (TEER) or decreases in sucrose permeability.32 The presence of bovine spongiform encephalopathy has limited the use of cow brains in Europe. However, Galla and coworkers have pioneered the use of PBMECs.33 Although these cells are not as well characterized as BBMECs, they do appear to have characteristics similar to those seen in BBMECs.4 Relevant transporters, such as P-gp and at least one MRP, are expressed in PBMECs.34,35 The procedures for isolating PBMECs are similar to those for isolating BBMECs.36,37 However, with PBMECs, cells are passaged once prior to use, and there is a report that addition of hydrocortisone to the medium results in a significant decrease in paracellular permeability.38 Although the literature on PBMECs is not as extensive as that on BBMECs, it is clear that PBMECs are suitable for pharmaceutical research. Although both BBMECs and PBMECs are widely accepted as suitable in vitro models of the BBB, there are some limitations to the utility of these models. Over the years, the in vitro cell culture models have proven to be fairly reliable predictors of BBB permeability. However, as with any in vitro model, confirmation of results using an animal model is recommended. One should also be aware that substrate-transporter interactions have been shown to be altered in allelic variants

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of the transporter P-gp.39 Thus, results need to be interpreted with the caveat that the transporters whose activity was assayed in the experiment may be qualitatively or quantitatively different from those in the human brain. Regulation of the transporters, along with any receptors that may be affected, may also be different in other species. A prime example of this is the pregnane-X receptor, which regulates the expression of P-gp and which has a very species-specific activation profile.40 BBMECs and PBMECs have been the models of choice for in vitro cell culture models of the BBB. However, it can be assumed that these models will be refined in the near future. One advance that may not be far off is the development of immortalized cell lines that are suitable for uptake and transport studies. Such cell lines would promote standardization of experimental conditions and would eliminate the need for the constant preparation of primary cells. Thus far, the immortalized cell lines that have been generated are not a suitable replacement for either BBMECs or PBMECs. One of the recurrent themes with immortalized cells is that their characteristics, especially with regard to transport, appear to change with increasing passage number.4 The generation of immortalized cell lines would allow new technologies to be employed in studies of transporters. For example, the overlap of transporter specificity with regard to substrates and inhibitors has been a major problem for researchers. Interfering RNA technology, which has the capability of knocking out either single or multiple genes with high specificity,41 could be used to determine transporter specificity. If immortalized cell lines could be produced from mice, it might be possible to take advantage of genetic knockout (KO) mice to generate cell lines that are genetically deficient in transporters. Clearly, the next few years will be an interesting time for those working with in vitro models of the BBB.

7.5. NASAL AND PULMONARY EPITHELIUM 7.5.1. The Respiratory Airway Epithelial Barrier Drug delivery by inhalation is attractive because of improved patient compliance and ease of administration compared to injection.42 In addition, macromolecules have shown surprisingly high absorption rates, particularly from the lower respiratory tract compared to other routes.43,44 As air passes through the nasopharyngeal region it is filtered, warmed, and humidified before entering the lungs. The trachea bifurcates into main bronchi, and branching continues through the small bronchi, bronchioles, terminal bronchioles, respiratory bronchioles, alveolar ducts, and the alveoli.45 The epithelial lining of the airways presents the primary barrier to drug transport throughout the airways; it is composed of a mix of ciliated and nonciliated pseudostratified columnar epithelial cells, secretory (mucous, goblet, serous, or Clara), and basal cells in the nasal, tracheal, and bronchial regions. Drugs and drug particles in the lowest portion of the respiratory tract, the alveolar region, encounter the thinnest cellular barrier, a large surface area for possible absorption and high blood flow.43,44

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Discussion of specific cell types characteristic of the epithelial cells along the respiratory tract and corresponding cell culture models for studying nasal and pulmonary drug transport are separated below, with generally more focus on the airway (tracheal and bronchial) epithelium and the alveolar epithelium.

7.5.2. The Nasal Epithelial Barrier and Cell Culture Models Upon intranasal administration, a drug is not as susceptible to dilution and first-pass effects as in oral delivery.46,47 The nasal route may also be an effective means of delivering drugs to the brain.46 Barriers to nasal delivery include the enzymes of the nasal mucosa, the epithelial barrier, the mucus layer, and limited absorption time resulting from mucociliary clearance.48 In primary culture of human nasal cells, the cells most relevant for transport studies are pseudostratified ciliated columnar epithelial cells from the medium and inferior turbinates.48 These cells show a high degree of differentiation, grow to a confluent monolayer in 6 to 8 days, and form tight junctions; however, they are expensive, their availability and life span are limited, and the model does not reflect the heterogeneity of cells in vivo.47–49 In a comparison of human nasal epithelial cells in primary culture with the RPMI 2650 and bovine turbinate cell lines, Werner and Kissel concluded that only the primary cultured cells appear suitable for drug transport studies because the cell lines lack differentiation and do not form a confluent monolayer with tight junctions.49 Another option for studying nasal transport is the use of excised mucosa of human or animal origin. Wadell and coworkers observed a good correlation for permeability data for seven of eight drugs tested in porcine nasal mucosa and the fraction absorbed upon nasal administration in humans.50 Excised mucosa have a lower TEER than primary cultured cells, which can make it difficult to distinguish between intact and damaged tissue; one must monitor tissue integrity and viability with alternative markers.47,48

7.5.3. The Airway Epithelial Barrier and Cell Culture Models Drug transport in the airway epithelium is by passive paracellular diffusion, transcytosis, and specific receptor binding.51 Only 4% of inhaled air is in the airway, which has less surface area available than the alveolar region, and airway cells have tighter junctions than alveolar cells; nevertheless, solute permeation in both types of cells is similar.43,51 The airway epithelium consists of ciliated, secretory (mucous, goblet, serous, or Clara), and basal cells. The ‘‘mucociliary escalator’’ serves to remove foreign material from the lungs. The epithelium thickness decreases from about 60 mm in the trachea to about 10 mm in the bronchioles, and the ciliated, pseudostratified cells more populous in the trachea and bronchi give way to an increasing number of secretory cells in the distal airways.43,51,52 Primary cell cultures of the airway epithelium are not as easy to maintain as cell lines, but they better represent the biological barrier. Primary cultured cells

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resemble in vivo cell layers in terms of tight junctions, apical cilia, and mucus production, but they are prone to infection and the quantity is often limited.43,51 The Calu-3 human submucosal gland cell line forms polarized cell monolayers with tight junctions, produces mucus, and develops apical cilia when grown at an air interface. Transport studies can be performed after 10–14 days in culture, and it has been shown that Calu-3 cells express P-gp and actively transport amino acids, nucleosides, and dipeptide analogs; organic anions, organic cations, polyamines, and efflux pump substrates are not actively transported.43,51,53–56 Because Calu-3 cells are not subject to the influence of multiple in vivo cell types, the expression of carrier proteins and enzymes may not reflect in vivo levels. Nevertheless, values obtained in Calu-3 permeability studies correlate well with those obtained from primary cultured rabbit tracheal epithelial cells and in vivo rat lung absorption studies.54 Mannitol permeation in Calu-3 cells is about 10 times less than that in vivo, but this is the same ratio difference between Caco-2 cells and in vivo intestinal epithelium.51 The 16HBE14o- and BEAS-2B cells are viral transformed bronchial cell lines.52,57 P-gp is expressed in 16HBE14o- cells, tight junctions form, and transport experiments can be carried out in 8 to 10 days, but the cells lack a representative mucus layer. Apical microvilli and cilia can be produced when 16HBE14o- cells are grown at an air interface, but this may not be favorable due to the basal origin of the cells.51,57 The use of BEAS-2B cells for transport studies is limited by their low TEER.57 7.5.4. The Alveolar Epithelial Barrier and Cell Culture Models In the separation of alveolar air space and blood circulation, the alveolar epithelium is a more restrictive paracellular barrier than the capillary endothelium. About onethird of the alveolar epithelial cells are type I cells, but these cells make up approximately 95% of the cellular surface area. The remaining two-thirds of the alveolar epithelial cells that comprise the remaining 5% of the cellular surface area are the surfactant-producing cuboidal type II cells.43,45,52 Type I cells have thin cytoplasmic extensions and exhibit a large number of plasmalemmal invaginations called ‘‘caveolae,’’ which may play a role in macromolecular and protein transport across the ‘‘blood-air barrier’’ of the lung.44,45,58,59 Although interspecies differences can be circumvented through the use of primary cultures of human alveolar epithelial cells, routine use of these cells is limited by availability.58,60 Primary culture of rat type II alveolar epithelial cells is economical and demonstrates high reproducibility, and its phospholipid secretion is similar to that of human type II cells.43 P-gp is expressed in both human and rat type I cells but not in freshly isolated rat type II cells.61 It is assumed that alveolar type II cells are progenitors for regenerating type I cells in vivo, and type II cells in culture lose their cuboidal appearance, lamellar bodies, and microvilli, and the number of surfactant proteins decrease. Monolayers are formed in 5 to 8 days, and the transdifferentiation to type I-like cells is complete within 7–8 days, characterized by the development of attenuations, tighter junctions, and increasing expression of

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P-gp.43,45,61 If necessary, cell culture conditions can be controlled to maintain the type II cell phenotype.60 The A549 alveolar epithelial cell line exhibits metabolic and transport properties consistent with those of type II cells in vivo. A549 cells have microvilli and lamellar bodies and synthesize phospholipids. However, A549 cells do not form domes like type II cells, they do not differentiate into type I-like cells, and they do not form tight junctions.42,43,45,62 Because no appreciable TEER is observed in the A549 cells, performing transport studies with low molecular weight drugs and xenobiotics is not feasible.62 7.6. THE OCULAR EPITHELIAL AND ENDOTHELIAL BARRIERS 7.6.1. The Corneal Epithelial Barrier and Cell Culture Models The anterior ocular barrier consists of two distinct components—the cornea and conjunctiva. The anterior surface of the eyeball is formed by the optically clear cornea and is composed of multiple cellular and acellular layers. However, it is the corneal epithelium on the surface of the eye that generally forms the primary barrier to drug delivery to pharmacological targets within the eye. The stratified corneal epithelium has tight intercellular junctions between the cells of the outermost layer and restricts drug diffusion to the transcorneal epithelial route. Transcorneal epithelial passage of drugs is largely dependent on their lipophilic nature. Therefore, the mechanism for drug distribution across the cornea is considered to be purely passive diffusion.63 The current method for culturing corneal epithelial cells is based on that developed by McPherson et al. in the 1950s while studying the cellular nutritional requirements for the preservation of corneal tissues.64 Primary corneal epithelial cells from both humans and rabbits have been successfully cultured.65–69 The advantage of the primary culture model is that the tissue has recently been removed from an in vivo condition and thus more closely resembles the structure and function of the epithelium in vivo. The disadvantage is that these cells degrade quickly in vitro as a result of the physical removal of the cells, as well as the constantly changing environment. Corneal epithelial cells have been immortalized in a variety of ways.70–76 Results obtained with immortalized cells are highly variable and cell-type dependent; the expression of different phenotypes is altered or absent altogether. Thus, primary lines propagated by extended subculture have been more successful.77–82 The latter lines are advantageous in that they very closely resemble normal, freshly excised cells. A newer, more useful culture model incorporates an air-interface condition.81 The conjunctiva provides a mucosal covering for the eye and is composed of a pseudostratified columnar epithelium. The conjunctiva generally has a higher permeability to hydrophilic compounds than does the cornea, and is also more permeable to macromolecules due to the larger and more numerous paracellular spaces.83,84 Passage of drugs across the conjunctiva into the systemic circulation is generally considered nonproductive absorption because delivery to pharmacological sites in the eye (i.e., within the aqueous humor) does not occur.63 Primary

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conjunctival cells from rabbits have been cultured. However, these cells lose viability after only three passages.63 Despite these disadvantages, primary conjunctival cells have proven useful for studying drug transport in liquid-covered cultures (LCC)85 as well as in air-interface cultures (AIC).86,87 Corneal and conjunctival cell cultures both exhibit some potential for application in drug transport studies. However, neither cell culture system has been extensively exploited for this purpose.63 7.6.2. The Blood-Retinal Epithelial Barrier and Cell Culture Models Accessibility of drugs to the posterior portion of the eye is necessary to treat a number of autoimmune and inflammatory diseases associated especially with aging. As a consequence, drug delivery across the blood-retinal barrier is important to the pharmaceutical chemist. The blood-retinal barrier (BRB) limits the passage of materials between the systemic circulation and the retina88 and is formed by the tight junctions of a monolayer of retinal pigment epithelium (RPE). The epithelium comprises the outer portion of the BRB, while the retinal vascular endothelium (RVE) makes up the inner portion.89 The tight junctions of the RPE retard diffusion between the RPE cells, delineate the apical and basolateral membrane domains, and maintain the surface specific distribution of the RPE membrane proteins. The asymmetric nature of the RPE is essential for the transport of nutrients and the regulation of fluid flow between the neural retina and the fenestrated capillaries of the choroids.88 This outer barrier also prevents the paracellular passage of ions and metabolites that escape the choriocapillaris.80–93 Thus, the RPE is strategically located within the retina and serves as a model with high interest. The RVE is very comparable to the BBB.94,95 There are several ways to purify a primary RPE culture.88,96–100 The process of isolation, culture technique, and cryopreservation is well documented.98,99 Grown on porous supports, RPE cells have been applied in a limited number of permeability and toxicity studies.88 The isolation and culture technique of primary RVE cells from Macaca monkeys is also documented;101 however, their application in drug permeability studies has been minimal.88

7.7. THE PLACENTAL BARRIER 7.7.1. The Syncytiotrophoblast Barrier The placenta separates maternal and fetal circulations with the primary function of allowing and promoting appropriate metabolic exchange.102 Appropriate placental function is very important for two reasons. First, respiratory gases and nutrients necessary for fetal growth are transported from the maternal circulation across the placenta to the fetus. Second, the metabolic waste products from the fetus enter the maternal circulation through the placenta. This exchange can occur by both passive diffusion and active transport involving transport proteins. Nutrients,

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drugs, and xenobiotics require the presence of specific transporters for their transfer.103 The human placental barrier is unique among mammalian species and is composed simply of a single layer of multinucleated cells, the syncytiotrophoblasts. The syncytial trophoblast forms the rate-limiting barrier for drug permeation across the human placenta and between maternal and fetal compartments.103 Asymmetry in the distribution in the expression of transporters in the syncytiotrophoblast results in polarized transport of substrates. As such, transporters specific for transport of substrates from the maternal circulation to the fetal circulation are expressed in the maternal facing brush border membrane (e.g., serotonin, norepinephrine, carnitine, monocarboxylate, folate, and P-gp transporters). Likewise, the fetal facing surface has a distinct array of transporters (e.g., extraneuronal monoamine transporter).104,105 7.7.2. The Syncytiotrophoblast Barrier and Cell Culture Models By comparison with other tissues discussed in this review, the identification of representative cell culture models of the placental barrier for drug delivery applications is more recent. Prior to the development of cell culture techniques, isolated tissues and cells of human placenta were used; these methods have been described elsewhere.102 Recent advances in cell and tissue culture techniques provide the potential for evaluation of drug transport or metabolism processes at the placenta. Techniques are available for culturing trophoblasts of both animal and human origin.106 However, our focus here is primarily on human systems. Primary explant and isolated cell cultures of human cytotrophoblasts have been well described;106–109 however, these systems do not form confluent monolayer systems adequate for transcellular transport studies.105 Three cell lines (BeWo, JAr, and JEG) have been generated from malignant trophoblastic cells and are available from the American Tissue Culture Collection (ATCC). The BeWo cell line, which is heterogeneous by several criteria,110 is comprised of cytotrophoblasts with minimal differentiation to syncytium111 under nonactivated conditions.112 This is in contrast to primary cultures of term human cytotrophoblasts, which spontaneously aggregate and form syncytia.107 The JEG3 cell line was originally derived from BeWo.113 It expresses human chorionic gonadotrophin and placental lactogen, a characteristic of normal trophoblasts,114 and forms large multinucleated syncytia in culture similar to syncytiotrophoblasts in vivo.115 The JAr cells synthesize human chorionic gonadotrophin and steroids, characteristic of early placental trophoblasts,116 and also have the ability to differentiate into syncytiotrophoblast-like cells in vitro.117 While the JEG3 and JAr cells are adequate for drug uptake studies, only a clone of the BeWo cell line forms a confluent monolayer suitable for transcellular drug delivery studies and has been applied to study a variety of nutrient and metabolic pathways in the trophoblast. The BeWo cell line has also proven useful in characterizing drug transport and drug efflux mechanisms present in the normal trophoblast.106,118,119

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7.8. THE RENAL EPITHELIUM 7.8.1. The Renal Epithelial Barrier Nephrons are the functional units of the kidney. Each nephron consists of a filtering component, the glomerulus, and a tubular portion made up of three distinct segments—proximal tubule, Henle’s loop, and distal convoluted tubule. The net renal elimination is a sum of three distinct mechanisms: glomerular filtration, tubular reabsorption, and tubular secretion. Glomerular filtration consists of passive removal of substances of molecular weight less than 5 kDa from the blood. This forms the ultrafiltrate, which contains glucose, amino acids, vitamins, and proteins. The ultrafiltrate then passes into the lumen of the tubular portion.120 The tubule is made up of a single layer of epithelial cells resting on a basement membrane. The apical side of the cell layer faces the lumen of the tubule, while the basolateral surface lies adjacent to the blood vessel. In the tubular portion, nutrients like glucose, proteins, vitamins, and amino acids, ions, and water are salvaged from the ultrafiltrate and are either transported across the tubular epithelium into blood or metabolized in the cells. Waste products on the blood side are taken up the tubule and secreted into the lumen to be eliminated in the urine. The transport across the epithelial cells occurs either by passive diffusion or by active carrier-mediated transport and can include a number of drugs.120–124 Various in vivo and in vitro models have been described to study the elimination of drugs from the kidneys. Glomerular filtration and clearance rates require in vivo studies or are performed on perfused whole kidneys.121,122 Tubular secretion and reabsorption of substances have been studied in kidney slices, tubular segments, tubular suspensions, primary cultures, and established cell lines.

7.8.2. Renal Epithelial Cell Culture Models Isolation and culture of primary cells from tubular segments of the nephron from different species such as rabbit, rat, flounder, and human have been described.125–129 Although the methods differ, they basically involve homogenization of the organ followed by protease treatment to dissociate cells. The major drawback of primary cultures is that cell types other than the intended one are also copurified. In the case of renal epithelium, fibroblasts are major contaminants; as these grow at a faster rate than the epithelial cells, they form the majority population in the culture. Taub et al. have described a defined medium containing hormones and epithelial cell growth additives which, when used in place of serum, permit selective growth of only the epithelial cells.125 Primary cultures retain the differentiated properties of the tissues of origin. The continuous cell lines produce a homogeneous population of cells and are easier to work with in general. Therefore, a number of renal epithelial cell lines have been developed. Two of the more widely used and well-characterized cell lines are MDCK and LLC-PK1. MDCK cells were derived from the distal tubule of dog kidney, while LLC-PK1 cells originated from the proximal tubule of the pig

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kidney.18,19 One of the critical requirements for a cell line to be useful for drug transport studies is that the cells form a polarized monolayer with respect to expression and function of transporters and enzymes when grown onto a microporous membrane. Both MDCK and LLC-PK1 cells form well-defined junction structures at confluency, as well as exhibit a significant TEER and a measurable potential difference.18,130 In comparison to MDCK cells, LLC-PK1 cells express higher activities of proximal tubular brush-border membrane enzymes—gamma-glutamyltranspeptidase, alkaline phosphatase, and leucine aminopeptidase as well as lysosomal enzymes—acid phosphatase and N-acetyl glucosaminidase. Conversely, the distal tubular membrane marker Naþ-Kþ ATPase shows higher activity in MDCK cells.131 Many transporters display overlapping substrate specificity and, as most cells express a number of different transporters, it is difficult to evaluate the specific interactions between a given drug and transporter. Overexpression of these proteins in cell lines is a way to study the substrate specificity and transport characteristics. MDCK and LLC-PK1 cells, due to their polarized nature, are capable of sorting membrane proteins to either the apical (seen with influenza viral proteins) or basolateral surface (vesicular stomatitis viral proteins).132 Human and rat multidrug resistance–associated protein (MRP-2) are located on the apical surface and, when expressed in MDCK cells, they are predominantly routed to the luminal membrane.133 When expressed in HEK cells these proteins were localized in intracellular membranes, but they do retain full functional activity. The apical transporter P-gp, when expressed in MDCK cells, is trafficked to the luminal membrane.134 Overexpression of canalicular multispecific organic anion transporter (cMOAT, also known as MRP2) in MDCK cells shows that it can transport organic anions S-(2,4-dinitrophenyl)-glutathione and S- (PGA1)-glutathione, compounds which were thought to be specific for MRP1. The transport of these compounds is not completely abolished in the presence of inhibitors specific to MRP1.135 MDCK cells overexpressing P-gp and canalicular multispecific organic anion transporter have been shown to transport the anticancer drug etopside.136 Although MDCK cells are easily transfected with specific transporters, their widespread use for high-throughput screening may be subject to some limitations due to cell type– dependent expression and functional differences.137–139

7.9. CONCLUSIONS Current technologies provide for availability of cell culture models from a selection of the significant tissue barriers as either primary cell culture systems or continuous cell line cultures. A number of endothelial and epithelial cell systems can now be grown onto permeable supports as monolayer systems to facilitate transcellular transport. These cell culture models provide powerful tools for the pharmaceutical chemist to characterize fundamental cellular transport mechanisms at the biochemical and molecular levels and effective screening systems to facilitate appropriate drug design and development. Moreover, they all have applications in

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pharmacological and toxicological investigations. Expectations are that cell culture models or cell-based assays will continue to evolve into better representatives of in vivo tissues and, therefore, will be even more valuable to pharmaceutical scientists in more rapidly evaluating new chemical entities as potential drugs.

ACKNOWLEDGMENTS Work in the Audus laboratory described in this review has been supported in part by the National Institute on Child Health and Development (P01 HD39878), the National Cancer Institute (RO1 CA82801), and Boehringer Ingelheim Pharmaceutics.

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107. Kliman, H. J.; Nestler, J. E.; Sermasi, E.; Sanger, J. M.; Straus, J. F., 3rd. Endocrinology 1986, 118, 1567–1582. 108. Cimerikic, B.; Zamah, R.; Ahmed, M. S. Trophoblast Res. 1994, 8, 405–419. 109. Hall, C. S.; James, T. E.; Goodyear, C.; Branchard, C.; Guyda, H.; Giroud, C. J. P. Steroids 1977, 30, 569–580. 110. Aplin, J. D.; Sattar, A.; Mould, A. P. J. Cell Sci. 1992, 103, 435–444. 111. Pattillo, R. A.; Gey, G. O.; Delfs, E.; Mattingly, R. F. Science 1968, 159, 1467–1469. 112. Speeg, K. V., Jr.; Azizkhan, J. C.; Stromberg, K. Cancer Res. 1976, 36, 4570–4576. 113. Tuan, R. S.; Moore, C. J.; Brittingham, J. W.; Kirwin, J. J.; Atkins, R. E.; Wong, M. J. Cell Sci. 1991, 98, 333–342. 114. Kohler, P. O.; Bridson, W. E. J. Clin. Endocrinol. Metab. 1971, 32, 683–687. 115. Babalola, G. O.; Coutifaris, C.; Soto, E. A.; Kliman, H. J.; Shuman, H.; Straus, J. F., 3rd, Dev. Biol. 1990, 137, 100–108. 116. White, T. E.; Saltzman, R. A.; Di Sant’Agnese, P. A.; Keng, P. C.; Sutherland, R. M.; Miller, R. K. Placenta 1988, 9, 583–598. 117. Grummer, R.; Hohn, H. P.; Mareel, M. M.; Deker, H. W. Placenta 1994, 15, 411–429. 118. Young, A. M.; Fukuhara, A.; Audus, K. L. In Cell Culture Models of Biological Barriers; Lehr, C.-M., Ed. Taylor & Francis: New York, 2002, pp. 337–349. 119. Young, A. M.; Allen, C. E.; Audus, K. L. Adv. Drug Deliv. Rev. 2003, 55, 125–132. 120. Vander, A. J. In Renal Physiology; McGraw-Hill: New York, 1995, 121. Bonate, P. L.; Reith, K.; Weir, S. Clin. Pharmacokinet. 1998, 34, 375–404. 122. Zhang, L.; Brett, C. M.; Giacomini, K. M. Annu. Rev. Pharmacol. Toxicol. 1998, 38, 431–460. 123. Inui, K.; Masuda, S.; Saito, H. Kidney Int. 2000, 58, 944–958. 124. Van Aubel, R. A.; Masereeuw, R.; Russel, G. Am. J. Physiol. Renal Physiol. 2000, 279, F216–F232. 125. Chung, S. D.; Alavi, N.; Livingston, D.; Hiller, S.; Taub, M. J. Cell Biol. 1982, 95, 118–126. 126. Sakhrani, L. M.; Fine, L. G. Miner Electrolyte Metabol. 1983, 9, 276–281 127. Wilson, P. D.; Dillingham, M. A.; Breckon, R.; Anderson, R. J. Am. J. Physiol. 1985,248, F436–F443. 128. Dickman, K. G.; Renfro, J. L. Am. J. Physiol. 1986, 251, F424–F432. 129. Elliget, K. A.; Trump, B. F. In Vitro Cell Dev. Biol. 1991, 27A, 739–748. 130. Misfeldt, D. S.; Hamamoto, S. T.; Pitelka, D. R. Proc. Natl. Acad. Sci. USA 1976, 73, 1212–1216. 131. Gstraunthaler, G.; Pfaller, W.; Kotanko, P. Am. J. Physiol. 1985, 248, F536–F544. 132. Boulan, E. R.; Sabatini, D. Proc. Natl. Acad. Sci. USA 1978, 75, 5071–5075. 133. Cui, Y.; Konig, J.; Buchholz, J. K.; Spring, H.; Leier, I.; Keppler, D. Mol. Pharmacol. 1999, 55, 929–937. 134. Pastan, I.; Gottesman, M. M.; Ueda, K.; Lovelace, E.; Rutherford, A. V.; Willingham, M. C. Proc. Natl. Acad. Sci. USA 1988, 85, 4486–4490. 135. Evers, R.; Kool, M.; van Deemter, L.; Janssen, H.; Calafat, J.; Oomen, L. C.; Paulusma, C. C.; Oude Elferink, R. P.; Baas, F.; Schinkel, A. H.; Borst, P. J. Clin. Invest. 1998, 101, 1310–1319.

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136. Guo, A.; Marinaro, W.; Hu, P.; Sinko, P. J. Drug Metab. Dispos. 2002, 30, 457–463. 137. Putnam, W. S.; Ramanathan, S.; Pan, L.; Takahashi, L. H.; Benet, L. Z. J. Pharm. Sci. 2002, 91, 2622–2635. 138. Tang, F.; Horie, K.; Borchardt, R. T. Pharm. Res. 2002, 19, 765–772. 139. Tang, F.; Horie, K.; Borchardt, R. T. Pharm. Res. 2002, 19, 773–779.

8 PRODRUG APPROACHES TO DRUG DELIVERY LONGQIN HU Department of Pharmaceutical Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854

8.1. Introduction 8.2. Basic concepts: definition and applications 8.2.1. Increasing lipophilicity to increase systemic bioavailability 8.2.2. Sustained-release prodrug systems 8.2.3. Improving gastrointestinal tolerance 8.2.4. Improving taste 8.2.5. Diminishing gastrointestinal absorption 8.2.6. Increasing water solubility 8.2.7. Tissue targeting and activation at the site of action 8.2.7.1. Tumor hypoxia and bioreductive activation of anticancer prodrugs 8.2.7.2. Activation of prodrugs by tissue- or tumor-specific enzymes 8.2.7.3. Antibody- or gene-directed enzyme prodrug therapy 8.2.7.4. Tumor-specific transporters 8.3. Prodrug design considerations 8.4. Prodrugs of various functional groups 8.4.1. Ester prodrugs of compounds containing

COOH or

OH 8.4.2. Prodrugs of compounds containing amides, imides, and other acidic NH 8.4.2.1. Mannich bases 8.4.2.2. N-a-Acyloxyalkyl derivatives 8.4.3. Prodrugs of amines 8.4.3.1. Amides 8.4.3.2. N-a-Acyloxyalkoxycarbonyl derivatives 8.4.3.3. N-Mannich bases

Drug Delivery: Principles and Applications Edited by Binghe Wang, Teruna Siahaan, and Richard Soltero ISBN 0-471-47489-4 # 2005 John Wiley & Sons, Inc. 125

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PRODRUG APPROACHES TO DRUG DELIVERY

8.4.3.4. Azo prodrugs 8.4.3.5. Schiff base prodrugs 8.4.4. Prodrugs for compounds containing carbonyl groups 8.4.4.1. Schiff bases and oximes 8.4.4.2. Enol esters 8.4.4.3. Oxazolidines 8.4.4.4. Thiazolidines 8.5. Drug release and activation mechanisms 8.5.1. Cascade release facilitated by linear autodegradation reactions 8.5.2. Cascade release facilitated by intramolecular cyclization reactions 8.5.3. Cascade activation through intramolecular cyclization to form cyclic drugs 8.6. Prodrugs and intellectual property rights—two court cases References

8.1. INTRODUCTION Most drugs, in order to produce their desired pharmacological action, have to overcome many hurdles before reaching the desired site of action. These hurdles include the intestinal barrier, the blood-brain barrier (BBB), and metabolic reactions that could render them inactive. These three subjects are covered in Chapters 2, 3, and 6, respectively, and therefore will not be discussed in detail here. Most drugs distribute randomly throughout the body, and the amount of drugs reaching the site of action is relatively small. For an effective amount to reach the site of action and not cause severe systemic side effects, a drug must possess certain physicochemical properties that make it conducive to penetration through various biological membranes (i.e., sufficiently bioavailable), to avoid metabolic inactivation by various enzymes, and to avoid retention in body depot tissues that could lead to undesirable long-lasting effects. These desired physicochemical properties are not always present in pharmacologically active compounds. With the advance of new technologies such as combinatorial and computational chemistry, more and more compounds are being identified with extremely potent in vitro activity but are found to be inactive in vivo. They may have the optimal configuration and conformation needed to interact with their target receptor or enzyme, but they do not necessarily possess the best molecular form and physicochemical properties needed for their delivery to the site of action. Some of the problems often encountered include (1) limited solubility and poor chemical stability preventing the drug from being adequately formulated, (2) low or variable bioavailability due to incomplete absorption across biological membranes or extensive first-pass metabolism, and (3) lack of site specificity. Further structural modifications are often performed but do not always solve all the problems. Another approach that

BASIC CONCEPTS: DEFINITION AND APPLICATIONS

127

is often effective in solving some of these delivery problems is the design of prodrugs by attaching a promoiety to the active drug.1–3 This chapter will focus on the various prodrug approaches that have been used to overcome many of the pharmaceutical and pharmacokinetic barriers that hinder optimal delivery of the active drug.

8.2. BASIC CONCEPTS: DEFINITION AND APPLICATIONS A prodrug by definition is inactive or much less active and has to be converted to the active drug within the biological system. There are a variety of mechanisms by which a prodrug can be activated. These include metabolic activation mediated by enzymes present in the biological system as well as the less common, simple chemical means of activation such as hydrolysis. Prodrugs occur in nature. One example is proinsulin, which is synthesized in the pancreas and releases its active moiety, insulin, and an inactive peptide. Codeine is another example; it can be regarded as a prodrug of morphine, which is responsible for its analgesic effect. Most synthetic prodrugs are prepared by attachment of the active drug through a metabolically labile linkage to another molecule, the ‘‘promoiety’’. The promoiety is not necessary for activity but may impart some desirable properties to the drug, such as increased lipid or water solubility or site specificity. Advantages that can be gained with such a prodrug include increased bioavailability, alleviation of pain at the site of injection, elimination of an unpleasant taste, decreased toxicity, decreased metabolic inactivation, increased chemical stability, and prolonged or shortened duration of action. 8.2.1. Increasing Lipophilicity to Increase Systemic Bioavailability This is the most successful application of prodrugs. Because of the lipid bilayer nature of biological membranes, the rate of passive drug transport is affected by both lipophilicity and aqueous solubility (also called ‘‘hydrophilicity’’). The rate of passive diffusion across the biological membrane will increase exponentially with increasing lipophilicity and then level off at higher lipophilicity. This is due to the fact that an increase in lipophilicity is usually accompanied by a decrease in water solubility and will eventually decrease the flux over the membrane due to poor water solubility. The design of prodrugs aims to achieve a balance between lipophilicity and aqueous solubility in order to improve passive drug transport across various biological membranes. Using data drawn from U.S. Adopted Names, the World Drug Lists, and Pfizer internal compound collections, it was concluded that, to have good membrane permeability, drugs should have a relatively low molecular weight (500), be relatively nonpolar, and partition between an aqueous and a lipid phase in favor of the lipid phase but, at the same time, possess certain water solubility ð
1  LogP  þ5Þ.4 The majority of effective oral drugs obey this socalled Lipinski’s rule of 5.

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PRODRUG APPROACHES TO DRUG DELIVERY

Since most drugs are either weak acids or weak bases, they are often given and present in the salt form under relevant physiological conditions. Therefore, dissociation constants also affect membrane permeability, and thus bioavailability. It is generally accepted that the neutral, unionized and thus most lipophilic form of an acidic or basic drug is absorbed far more efficiently than the ionized species. In these cases, the distribution between the ionized and neutral form depends strongly on pH. The effective partition coefficient for a dissociative system (LogD) gives the correct description of such complex partitioning equilibria. LogD ¼ LogPHA
logð1 þ 10ðpH
pKaÞ Þ

for an acid

LogD ¼ LogPB
logð1 þ 10ðpKa
pHÞ Þ

for a base

where PHA and PB are the intrinsic partition coefficients of the weak acid and weak base, respectively. Programs such as ACD/LogP and cLogP are available to calculate with reasonable accuracy the LogP and LogD values using a structure-fragment approach as well as internal structure databases. To illustrate the principles discussed in this chapter, examples will be given with their LogP and/ or LogD values calculated using the Advanced Chemistry Development (ACD) Software. Many prodrugs feature the addition of a hydrophobic group in order to increase their lipid solubility to improve their gastrointestinal absorption. Bacampicillin (2), pivampicillin (3), and talampicillin (4) are more lipophilic esters of ampicillin (1), and pivmecillinam (6) is a more lipophilic ester prodrug of mecillinam (5), all with improved oral bioavailability. For example, absolute oral bioavailability in horses was 39%, 31%, and 23% for bacampicillin, pivampicillin, and talampicillin, respectively, compared to only 2% for ampicillin sodium.5 Esterification of carboxylic acid in ampicillin (1) resulted in an increase of 0.8-1.4 unit in LogP. More significant are the increases in LogD values for the prodrugs when ionization of the amino group is taken into consideration; as much as a 4-unit difference in LogD is estimated at pH values in the intestines where the prodrugs are believed to be absorbed. Other prodrugs of antibiotics include esters of carbenicillin (for urinary tract infection), cefotiam, and erythromycin.

NH2 H H H N S O

O

N O

N

N O

(1) Ampicillin (2) Bacampicillin (3) Pivampicillin

H H

O

R (4) Talampicillin

ACD/LogD pH 7 pH 1

R=H R = -CH(CH3)OCOOC2H5 R = -CH(CH3)OCOC(CH3)3

1.350±0.320 2.172±0.894 2.552±0.884

–1.54 1.99 2.37

–1.72 –0.93 –0.55

R=

2.789±0.882

2.61

–0.31

1.493±0.866 3.453±0.921

–1.01 1.77

–1.58 0.35

O

O

S

N O

ACD/LogP

O

R

(5) Mecillinam R=H (6) Pivmecillinam R = -CH(CH3)OCOC(CH3)3

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BASIC CONCEPTS: DEFINITION AND APPLICATIONS

Enalapril (8) is an ester prodrug of enalaprilat (7). The latter binds tightly to the angiotensin-converting enzyme (ACE) but is transported with low efficacy by the peptide carrier in the gastrointestinal tract. The prodrug enalapril has a higher affinity for the peptide carrier6 and is much better absorbed, with about 60% oral bioavailability.7,8 As a matter of fact, all ACE inhibitors except captopril and lisinopril are administered as prodrugs; other commercialized ACE inhibitor prodrugs include perindopril, quinapril, ramipril, cilazapril, benazepril, spirapril, and trandolapril, all based on esterification of the same carboxylic acid group.9 The esters are hydrolyzed in vivo, after absorption, to the corresponding active but poorly absorbed dicarboxylate forms. O RO

ACD/LogP N H

N O

O

OH

(7) Enalaprilat (8) Enalapril

R=H R = Et

2.102±0.574 2.983±0.580

ACD/LogD pH 7 pH 1 –1.45 –0.12

–0.92 –0.10

Valacyclovir (10)10,11 and famciclovir (12) are ester prodrugs of acyclovir (9) and penciclovir (11), respectively, for the treatment of viral infections. Both acyclovir and penciclovir exhibit site-specific conversion to the active triphosphate species by viral thymidine kinase. They show remarkable antiviral selectivity and specificity. However, their oral bioavailability is quite low, 15–20% of an oral dose being absorbed in humans for acyclovir and 5% for penciclovir.12 Both valacyclovir and famciclovir have no intrinsic antiviral activity, and both are rapidly hydrolyzed to acyclovir and penciclovir by esterases present in the liver and gut wall. Valacyclovir displays a mean absolute bioavailability of 54%, a threefold increase in oral bioavailability over acyclovir, while famciclovir has an absolute bioavailability of 77% in humans.13–15 Famciclovir’s better bioavailability could be explained by the increase in lipophilicity; the high oral bioavailability of valacyclovir was also partly attributed to the involvement of an active transport mechanism through PEPT1.16 Therefore, in addition to increasing lipophilicity, prodrug design can utilize active transport mechanisms as a means of enhancing bioavailability. O N

HN H2N

ACD/LogP

N

N O

ACD/LogD pH 7 pH 1

(9) Acyclovir R=H (10) Valacyclovir R = L-Valyl-

–1.760±0.489 0.040±0.577

–1.76 –0.78

–3.76 –4.76

(11) Penciclovir (12) Famciclovir

–2.031±0.584 –0.088±0.265

–2.03 –0.09

–4.44 –3.27

OR

O N

HN H2N

N

N

OR OR

R=H R = CH3CO-

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PRODRUG APPROACHES TO DRUG DELIVERY

8.2.2. Sustained-Release Prodrug Systems Antipsychotic drugs are the mainstay treatment for schizophrenia and similar psychotic disorders. Long-acting depot injections of antipsychotic drugs are extensively used as a means of long-term maintenance treatment. The duration of action for many antipsychotic drugs with a free hydroxyl group can be considerably prolonged by the preparation of long-chain fatty acid esters with very high LogP values (usually 7 or above). Fluphenazine enanthate (14) and fluphenazine decanoate (15) were the first of these esters to appear in clinical use and are longer-acting, with fewer side effects than the parent drug. The ability to treat patients with a single intramuscular injection every 1–2 weeks with the enanthate or every 2–3 weeks with the decanoate esters means that problems associated with patient compliance with the drug regimens and with drug malabsorption can be reduced.17 Esterification of antipsychotic drugs with decanoic acid yields very lipophilic prodrugs which are dissolved in a light vegetable oil such as Viscoleo or sesame oil. Intramuscular injection creates an oily depot from which the prodrug molecules slowly diffuse into the systemic circulation, where they are hydrolyzed quickly by esterases to the active moieties. These depot forms allow these drugs to be given only once or twice a month, permitting the long-term treatment of schizophrenia. Antipsychotic drugs available in depot formulation include fluphenazine (13), flupenthixol, haloperidol, and zuclopenthixol in their enanthate or decanoate esters. ACD/LogP

S CF3

N

(13) Fluphenazine R= H (14) Fluphenazine enanthate R = -CO(CH2)5CH3 (15) Fluphenazine decanoate R = -CO(CH2)8CH3

4.841±0.460 8.034±0.453 9.628±0.453

N N

O

R

Anabolic steroids such as nandrolone and testosterone, anti-inflammatory glucocorticoids such as methylprednisolone, and contraceptives such as estradiol and levonorgestrel all have slow-release formulations of their ester prodrugs in the market. 8.2.3. Improving Gastrointestinal Tolerance Temporary masking of carboxylic acid groups in nonsteroidal anti-inflammatory drugs was proposed as a promising means of reducing gastrointestinal toxicity resulting from direct mucosal contact mechanisms. Morpholinoalkyl esters (17 and 19, HC1 salts) of naproxen (16) and indomethacin (18) were evaluated in vitro and in vivo for their potential use as prodrugs for oral delivery.18 The prodrugs were freely soluble in simulated gastric fluid and pH 7.4 phosphate buffer and showed a minimum of a 2000-fold increase in solubility over the parent drugs. The prodrugs were more lipophilic than the parent drugs and were quantitatively hydrolyzed to

131

BASIC CONCEPTS: DEFINITION AND APPLICATIONS

their respective parent drugs in vivo. The prodrugs were 30–36% more bioavailable orally than the parent drugs following a single dose in rats. They were significantly less irritating to gastric mucosa than the parent drugs following a single dose as well as chronic oral administration in rats. O O

MeO O

R

N

O

MeO

R

O Cl

(16) Naproxen (R = H) (17) R =

(CH2)n N

(18) Indomethacin (R = H) O

(19) R =

(CH2)n N

O

8.2.4. Improving Taste Oral drugs with a markedly bitter taste may lead to poor patient compliance if administered as a solution or syrup. The prodrug approach has been used to improve the taste of chloramphenicol (20), clindamycin, erythromycin, and metronidazole.19 A prodrug such as chloramphenicol palmitate (21), with LogP of around 10, does not dissolve in an appreciable amount in the mouth and, therefore, does not interact with the taste receptors. OH

O2N

H N

CCl2

O OR

ACD/LogP (20) Chloramphenicol (21) Chloramphenicol palmitate (22) Chloramphenicol sodium succinate

R=H R = -CO(CH2)14CH3 R = -COCH2CH2COO–Na+

1.018±0.321 9.920±0.756 2.287±0.849

ACD/LogD pH 7 pH 1 1.02 9.92 –0.34

1.02 9.92 2.29

8.2.5. Diminishing Gastrointestinal Absorption Many prodrugs have been evaluated in this context for colon-specific drug delivery. Colon targeting is of value for the topical treatment of diseases of the colon such as Crohn’s disease, ulcerative colitis, and colorectal cancer. Sustained colonic release of drugs can be useful in the treatment of nocturnal asthma, angina, and arthritis. Prodrugs have been designed to pass intact and unabsorbed from the upper gastrointestinal tract and undergo biotransformation in the colon, releasing the active drug

132

PRODRUG APPROACHES TO DRUG DELIVERY

molecule. Prodrug activation can be carried out by microflora and distinct enzymes present in the colon (such as azoreductase, glucuronidase, glycosidase, dextranase, esterase, nitroreductase, and cyclodextranase).20,21 Balsalazide (23), ipsalazide (24), olsalazine (25), and sulfasalazine (26) are azo-containing prodrugs developed for colon-specific delivery of an anti-inflammatory agent in the treatment of inflammatory bowel disease. As shown in Scheme 1, they can undergo azoreduction in the colon to release the active 5-aminosalicylic acid (5-ASA or mesalazine, 27). Other prodrugs evaluated for colon-specific delivery include conjugates of amino acids, glucuronide, glycoside, dextran, and cyclodextrin.22 O HO HO2C

NH (CH2)nCOOH N N (23) Balsalazide (n =2), (24) Ipsalazide (n=1) OH

HO HO2C

N N

azoreductase

CO2H

HO2C

O

O

HO2C

NH2

(27) Mesalazine (5-ASA)

(25) Olsalazine S

HO

HO

N H

N

N N (26) Sulfasalazine

Scheme 1

8.2.6. Increasing Water Solubility Poorly water-soluble, lipophilic drugs also have difficulty getting absorbed, as discussed earlier. The prodrug approach has been applied to circumvent solubility problems by introduction of an ionizable functional group such as phosphate esters, amino acid esters, and hemiesters of dicarboxylic acids, allowing various salts of such prodrugs to be formed. Prodrugs can also be used to increase water solubility in order to increase the amount of drug that will reach the systemic circulation through parenteral administration. Examples include chloramphenicol sodium succinate (22), hydrocortisone sodium succinate, methylprednisolone sodium succinate, betamethasone sodium phosphate, clindamycin phosphate, and prednisolone phosphate. In addition to the use of ionizable groups, disruption of the crystal lattice can also result in a significant increase in aqueous solubility, as illustrated by the antiviral agent vidarabine (28). The 50 -formate ester derivative (29) of vidarabine is 67-fold more soluble in water than vidarabine itself and has been attributed to

133

BASIC CONCEPTS: DEFINITION AND APPLICATIONS

disruption of the strong intermolecular interactions in the crystal, as indicated by the 85 C drop in the melting point.23 NH2 N

N RO

N OH O

N

ACD/LogP (28) Vidarabine (29) 5′-formate

R=H R = C-H

–1.458±0.470 –0.364±0.601

m.p. 260 °C 175 °C

solubility H2O (25 °C) 0.0018 M 0.12 M

O OH

8.2.7. Tissue Targeting and Activation at the Site of Action Prodrugs can be designed to target specific tissues. This is especially useful in improving the therapeutic effectiveness and decreasing the systemic toxicity of anticancer agents in the treatment of cancer. Anticancer agents are usually highly toxic, with a very small therapeutic index, and their therapeutic effectiveness is often limited by their dose-limiting side effects. Here, several strategies for targeting chemotherapeutic agents to cancers will be briefly discussed to illustrate the applications of prodrugs. For details, refer to Chapter 11 on metabolic activation and drug targeting. 8.2.7.1. Tumor Hypoxia and Bioreductive Activation of Anticancer Prodrugs Solid tumors often contain regions which are subject to chronic or transient deficiencies of blood flow and, therefore, to the development of chronic or acute hypoxia owing to the primitive state of tumor vasculature.24 Hypoxic cells in a solid tumor frequently constitute 10–20% and occasionally over half of the total viable tumor cell population. Agents that are active against proliferating cells are relatively ineffective against these hypoxic tumor cells, which are not actively replicating at the time of treatment but are capable of commencing proliferation at a later time and causing the tumor to regrow. Hypoxic cells also may be resistant to conventional chemotherapy due to pharmacodynamic considerations.24 To produce a therapeutic response, appropriate drug concentrations must be reached. Drugs that have physicochemical properties not conducive to diffusion into tumor tissue, or that are unstable or metabolized rapidly, may not reach chronically hypoxic tumor cells located in regions of severe vascular insufficiency. Therefore, the presence of hypoxic cells in solid tumors is an obstacle to effecting a cure. Since hypoxic cells located remotely from the vascular supply of a tumor mass may have a greater capacity for reductive reactions than their normal, welloxygenated counterparts, hypoxia could provide an opportunity for the design of selective cancer chemotherapeutic agents that could be reductively activated in these hypoxic cells.24 Several classes of agents are presently known which exhibit preferential cytotoxicity toward hypoxic cells through reductive activation. They

134

PRODRUG APPROACHES TO DRUG DELIVERY

include nitro compounds, quinones, and aromatic N-oxides.25,26 Examples include mitomycin C (30), CB1954 (31), EO9 (32), and AQ4N (33). O H2N

N

Me

NO2

CH2OCONH2 OMe NH

CONH2 O2N N

O (30) Mitomycin C

O

(31) CB1954

OH

OH

O

HN

OH

O

HN

N

O–

N

O

N H (32) EO9

OH

O– N

(33) AQ4N

8.2.7.2. Activation of Prodrugs by Tissue- or Tumor-Specific Enzymes Investigations of the biochemistry and molecular biology of cancer have also identified several reductive or proteolytic enzymes that are unique to tumors or tissues and could be used as potential therapeutic targets or prodrug-converting enzymes for novel cancer therapy. These include DT-diaphorase,27 prostate specific antigen (PSA),28 plasminogen activator,29 and members of matrix metalloproteinases.30 One such example is the peptide doxorubicin conjugate, glutaryl-Hyp-Ala-SerChg-Gln-Ser-Leu-Dox, L-377202 (34), which was reported to have the profile of physical and biological properties needed for further clinical development.31 Conjugate 34 was found to have a greater than 20-fold selectivity against PSA-secreting LNCaP cells relative to non-PSA-secreting DuPRO cells. In nude mouse xenograft studies, it reduced PSA levels by 95% and tumor weight by 87% at 21 mmol/kg, a dose below its maximal tolerated dose (MTD). On the basis of these results, this conjugate was selected for further studies in clinical trials to assess its ability to inhibit human prostate cancer cell growth and tumorigenesis. It was believed that PSA cleavage in and around prostate cancer cells would release, as shown in Scheme 2, dipeptide-doxorubicin conjugate (35), which would be further cleaved by aminopeptidases to the cytotoxic Leu-doxorubicin (36) and doxorubicin (37). 8.2.7.3. Antibody- or Gene-Directed Enzyme Prodrug Therapy Besides targeting hypoxic tumor cells and using tumor- or tissue-specific enzymes like PSA to activate prodrugs, other specific enzymes can be delivered to tumor tissues using antibodies or expressed by tumor cells through gene therapy and can be used as prodrug converting enzymes. These strategies are called ‘‘antibody-directed

135

BASIC CONCEPTS: DEFINITION AND APPLICATIONS O

OH

O OH OH

OMe O O HO

O

N O

N H

O

H N

N H OH

O

HO

H N

O

O Me OH NH H HO N O O

O N H

O

OH

PSA

CONH2 (34) L-377202 H-Ser-Leu-doxorubicin

H-Leu-doxorubicin

(35)

(36)

Doxorubicin indicates enzyme cleavage site

(37)

Scheme 2

enzyme prodrug therapy’’ (ADEPT) or ‘‘gene-directed enzyme prodrug therapy’’ (GDEPT). In these approaches, an enzyme is delivered site specifically by chemical conjugation or genetic fusion to a tumor-specific antibody or by enzyme gene delivery systems into tumor cells. This is followed by the administration of a prodrug, which is selectively activated by the delivered enzyme at the tumor cells. A number of these systems are in development and have been reviewed.32 Among the enzymes under evaluation is a bacterial nitroreductase from Escherichia coli. This is a flavin mononucleotide (FMN)–containing flavoprotein capable of reducing certain aromatic nitro groups to the corresponding amines or hydroxylamines in the presence of a cofactor NADH or NADPH. The nitroaromatics that were found to be good substrates of E. coli nitroreductase include dinitroaziridinylbenzamide CB1954 (31), dinitrobenzamide mustards SN 23862 (38), 4-nitrobenzylcarbamates (39), and nitrophenyl phosphoramides (40 and 41).33 NO2

NO2

O2N

CONH2 O2N

O

N Cl

R1

O Cl

(38) SN 23862

O

N H

Drug

(39) 4-nitrobenzylcarbamates

R2

N(CH2CH2Cl)2 P NH O

nitrophenyl phosphoramides (40) R1 = R2 = H (LH7) (41) R1, R2 = CH2CH2

136

PRODRUG APPROACHES TO DRUG DELIVERY

8.2.7.4. Tumor-Specific Transporters Antibody-drug conjugates would have to overcome problems inherent in proteins such as susceptibility to proteolytic cleavage and high immunogenicity; the latter could lead to an antibody response against the conjugate, thereby precluding further use. To increase the selectivity of chemotherapeutic agents, considerable efforts have also been made to identify biochemical characteristics unique to malignant tumor cells that could be exploited in a therapeutic intervention. The small and nonimmunogenic, tumor-specific molecules like folic acid are among the promising alternatives to antibody molecules as targeting agents for drug delivery. Folate conjugates of radiopharmaceuticals, magnetic resonance imaging (MRI) contrast agents, antisense oligonucleotides and ribozymes, proteins and protein toxins, immunotherapeutic agents, liposomes with entrapped drugs, and plasmids have all been successfully delivered to folate receptor–expressing cells.34 More details can be found in Chapter 9 on receptormediated endocytosis. 8.3. PRODRUG DESIGN CONSIDERATIONS Often medicinal chemists encounter a situation where a structure has adequate pharmacological activity but an inadequate pharmacokinetic profile (i.e., absorption, distribution, metabolism, and excretion). Prodrugs can be designed to improve physicochemical properties, resulting in improvement in pharmacokinetic as well as pharmaceutical properties. The pharmaceutical properties that could be improved, as discussed earlier, include drug product stability, taste and odor, pain on injection, and gastrointestinal irritation. These are great benefits that can be achieved through the design of prodrugs. However, regulatory issues should also be considered in the design process. In general, regulatory agencies are reluctant to register this type of product. Of particular concern is the fact that toxicological studies might not be relevant for human use of the drug because of differences in the rate and/or extent of formation of the active moiety—metabolic aspects. Experiments should thus be designed early to address these concerns. As examples of interspecies differences, the pivaloyloxyethyl ester of methyldopa was essentially hydrolyzed presystematically to pivalic acid and methyldopa at the same rate in human, dog, and rat, while the succinimidoethyl derivative was hydrolyzed faster in rat than in man and dog.35 This suggests that the succinimidoethyl ester of methyldopa was more resistant to extrahepatic esterase action in man and dog but not in rat. For different ester prodrugs of dyphylline, the relative rates of release were 1.3 to 13 times faster in rabbit plasma than in human plasma.36 The bond between the active moiety (parent drug) and the promoiety plays a major role in determining the pharmacokinetic properties of a prodrug. Knowledge about the nature of the bond and the promoiety may help explain the nature of the biotransformation process and its location in specific tissues or cells. The study of the fate in the body of the promoiety is particularly important from the safety point of view and should be investigated just as thoroughly as that of the active moiety. In some cases, the fate of the released carrier moiety is well known, such as the esters of methanol or ethanol; no extra study is needed during drug development. In other cases, additional pharmacokinetic investigations may be necessary.

PRODRUGS OF VARIOUS FUNCTIONAL GROUPS

137

Rational design of a prodrug should begin with identification of the problem(s) encountered with the delivery of the parent compound/drug and the physicochemical properties needed to overcome the delivery problem(s). Only then can the appropriate promoiety be selected to construct a prodrug with the proper physicochemical properties that can be effectively transformed to the active drug in the desired biological compartment. The most important requirement in prodrug design is naturally the adequate reconversion of the prodrug to the active drug in vivo at the intended compartment. This prodrug-drug conversion may take place before absorption (e.g., in the gastrointestinal system), during absorption (e.g., in the gastrointestinal wall or in the skin), after absorption, or at the specific site of drug action. It is important that the conversion be essentially complete because the intact prodrug, being usually inactive, represents unavailable drug. However, the rate of conversion would depend on the specific goal of the prodrug design. A prodrug designed to overcome poor solubility for an intravenous drug formulation should be converted very quickly to the active moiety after injection. If the objective of the prodrug is to produce sustained drug action through rate-limiting conversion, the rate of conversion should not be too fast. Prodrugs can be designed to use a variety of chemical and enzymatic reactions to achieve cleavage to generate their active drug at the desired rate and place. The design is often limited by the availability of a suitable functional group in the active drug for the attachment of a promoiety. Table 8.1 lists some of the common reversible prodrug forms for various functional groups that are often present in biologically active substances. The most common prodrugs are those that require hydrolytic cleavage, but reductive and oxidative reactions have also been used for the in vivo regeneration of the active drug. Besides using the various enzyme systems for the necessary activation of prodrugs, the buffered and relatively constant physiological pH may be used to trigger their release. Enzymes considered important to orally administered prodrugs are found in gastrointestinal walls, liver, and blood. In addition, enzyme systems present in the gut microflora may be important in metabolizing prodrugs before they reach the intestinal cells. In addition, site-specific delivery can be accomplished by exploiting enzymes that are present specifically or at high concentrations in the targeted tissues relative to nontarget tissues. A number of enzymes can also be delivered to targeted tissues through antibodies or gene-delivery approaches for the activation of subsequently administered prodrugs, as discussed earlier in this chapter. 8.4. PRODRUGS OF VARIOUS FUNCTIONAL GROUPS 8.4.1. Ester Prodrugs of Compounds Containing

COOH or

OH Due to the presence of a wide variety of esterases in various body tissues, it is not surprising that esters are the most common prodrugs used to improve gastrointestinal absorption. By appropriate esterification of molecules containing a carboxylic acid or hydroxyl group, it is possible to obtain derivatives with almost any desirable

138

PRODRUG APPROACHES TO DRUG DELIVERY

TABLE 8.1 Reversible Prodrug Forms for Various Functional Groups Present in Biologically Active Substances

Funtional Group O

Reversible Prodrug Forms O

O OH

R

R

O

O

O

O

O

R O

R′

O O

O R′

O

N H

R CH2OH

O O OH

O

R

S

O R

N

O O

N

OH On

X NH acidic

O

O

O

O

R′

R

R

R

R′ Ar

O

R O

N H

R′

O N

N

Ar Ar NO2

R′′

O

N

R

O R

OH

OR

O

S

OR′

N H

N H

O

O

N O

R O

O P

R

N

N O R

OH

O

R′

R N

S

S

R′

O

N H

R′

R

OH OH

P

O O

N H

R

R

O

O

O

N N

O

O

R

N H

R

R

S

NH2

O

O

O SH

R

P

O

R

O R P

O

O On

R

X

On X

N

N

O R

O P

R′

N

OH

O

O O

O

R′

X = C, n = 1 X = S, n = 2

R′

XH

X

NH2

N H

X = O or S

OH OH

O

N

O

R

hydrophilicity, lipophilicity, and in vivo lability. It should be noted that enzymecatalyzed ester hydrolysis is quite different from nonenzymatic ester hydrolysis in terms of electronic and steric requirements in the substrates. Enzymatic reactions are more likely influenced by steric rather than electronic effects. Experimental determination should be performed to evaluate the rate of cleavage under incubation with plasma or a homogenate from the intended tissue or organ where the prodrug would be activated. It should also be kept in mind that there are significant interspecies variations in the enzyme’s expression level and catalytic capacity.

139

PRODRUGS OF VARIOUS FUNCTIONAL GROUPS

O

O

Esterases

Drug

O

R

Drug

(42)

HO R Promoiety

O

Esterases

O

+

(43)

O R

OH

Drug

R

(44)

OH

+ HO Drug

Promoiety

(45)

Scheme 3

As shown in Scheme 3, esters in the form of 42 can be used as prodrugs for acid drugs (43), and the alcohol would serve as a promoiety. Esters in the form of 44 can be used as prodrugs for alcohol drugs (45), and here the acid would serve as a promoiety. Both the acyl and the alcohol portion surrounding the cleavable ester bond affect the enzyme-catalyzed ester hydrolysis. Sometimes because of steric hindrance in the active drug, direct ester formation with the existing functional group might not produce a prodrug that is sufficiently labile in vivo. This problem can be solved by designing the so-called cascade prodrugs containing double esters using a-acyloxyalkyl, carbonate, or alkoxycarbonyloxyalkyl esters (46, 47, or 48), where the terminal ester group is accessible for enzymatic cleavage (Scheme 4). Cascade prodrugs are those prodrugs that require a sequence of two or more reactions for drug release and activation, usually triggered by a first enzymatic-catalyzed reaction followed by a spontaneous chemical release/activation step(s). A number of such examples are known, including several prodrugs of b-lactam antibiotics, corticosteroids, and angiotensin II receptor antagonists. The 2-carboxylic acid on the thiazolidine ring of b-lactam antibiotics is required for antibacterial activity, O R′ R O Simple ester (42 or 44)

Bulky R

O R

R′′ O

Bulky R′

O O

O R

R′

α-Acyloxyalkyl ester (46)

O R

R′′ O

O

R′

Alkoxycarbonyloxyalkyl ester (48)

Scheme 4

O

R′

Carbonate ester (47)

O O

O

140

PRODRUG APPROACHES TO DRUG DELIVERY

providing an ideal site for attaching a promoiety in the design of ester prodrugs. But, because of steric hindrance, simple esters of this carboxylic acid group would resist enzymatic hydrolysis. Thus, a number of cascade prodrugs were made to extend the chain and render the terminal ester group easily accessible to hydrolytic enzymes. Examples of a-acyloxyalkyl ester prodrugs include bacampicillin (2), pivampicillin (3), pivmecillinam (6), and cefuroxime axetil (49). N

O

O

OMe H N

Et S

O

O O

N

O

HO

NH2

O O

O

O

O OEt

H

O H

O H

O

O (49) Cefuroxime axetil

(50) Prednicarbate

Prednicarbate (50) is an example of a carbonate prodrug of corticosteroids, while candesartan cilexetil (51) is a racemic mixture of an alkoxycarbonyloxyalkyl ester of candesartan (53) with a chiral center at the carbonate ester group. Following oral administration, candesartan cilexetil (51) undergoes rapid and complete hydrolysis during absorption from the gastrointestinal tract to form, as shown in Scheme 5, the O

O O

O

O O

O

OH

N N N N

HN

O

N N N

N

HN

N H2O

CO2 +

N

O

CH3CHO

HO

(52)

(51) Candesartan cilexetil ACD/LogP = 7.430±1.003

O OH N N N N

HN

O

(53) Candesartan ACD/LogP = 4.651±0.930 ACD/LogD = 0.54 at pH 7

Scheme 5

N

141

PRODRUGS OF VARIOUS FUNCTIONAL GROUPS

active drug candesartan (53), which is an achiral selective AT1 subtype angiotensin II receptor antagonist.37 For a more recent effort to find orally active aminomethyl-THF 1b-methylcarbapenems (Scheme 6), a number of mono and bis double esters were investigated as

O OH

OH S

N O

O

O

O

NH3

O

NH

tBut

S

N O

CO2

Et

O

O

O

O

Aminomethyl-THF1β-methylcarbapenems (54)

O

O

A bis double ester prodrug (55)

Scheme 6

potential prodrugs. The bis double ester derivatives such as (55) demonstrated enhanced oral activity, while the mono double ester derivatives did not demonstrate significantly improved oral activity due to the presence of a charged group.38 The a-acyloxylalkyl esters have also been extended to include the phosphate group, phosphonic acids, and phosphinic acids. One such example is fosinopril (57), an ACE inhibitor, where the phosphinic acid is O-a-acyloxyalkylated to increase lipophilicity to provide better absorption. ACD/LogD pH 7 pH 1

ACD/LogP (56) Fosinoprilat

O

P OR O

O

R=H O

N

(57) Fosinopril

R=

OH

Me

3.069±0.660

–1.93

3.06

5.810±0.679

2.65

5.81

Et

O Me

O-a-acyloxyalkyl ethers are also a useful prodrug type for compounds containing a phenol group. Such derivatives (58) are hydrolyzed by a sequential reaction involving formation of an unstable hemiacetal intermediate (59), as shown in Scheme 7. These kinds of ethers might be better prodrugs than normal phenol esters

R′′

Drug O

Enzymatic

O O

(58)

O

R′ HO

O (59)

R′

Scheme 7

Spontaneous

R′′

Drug

Drug OH

R′′

OH

CHO

(60)

142

PRODRUG APPROACHES TO DRUG DELIVERY

because they are more stable against chemical hydrolysis, but they are still susceptible to enzymatic hydrolysis by human plasma esterases. Carboxylic acids have also been masked as ketones and alcohols, which would require oxidation to convert to the active acid drugs. Nabumetone (61) is a nonacidic nonsteroidal anti-inflammatory prodrug (NSAID).39 After absorption, nabumetone undergoes extensive metabolism, the main circulating active form is 6-methoxy-2-naphthylacetic acid (62), a potent COX-2 inhibitor (Scheme 8). Since

O OH

Liver metabolism

MeO

O

MeO (61)

(62)

Scheme 8

nabumetone is not acidic and the active acid metabolite does not undergo enterohepatic circulation, nabumetone does not cause gastric irritation and is the most widely prescribed NSAID in the United States. 8.4.2. Prodrugs of Compounds Containing Amides, Imides, and Other Acidic NH 8.4.2.1. Mannich Bases Mannich base prodrugs could enhance the delivery of their parent drugs through the skin because of their enhanced water solubility as well as enhanced lipid solubility. N-Mannich bases, or N-acyl gem-diamines (67), are generally formed, as shown in Scheme 9, by reaction of an acidic NH com-

CH2O

HN R2

R1

HO

(63)

N R2 (64)

R1 O R

Chemical regeneration

Formation

(66)

R1 N R2 (65) NH2

O R

N H

N-Mannich base or N-acyl gem-diamine

N R2 H+

O R

N H

R1

(67) B H R1 N R2 (68) BH+

Scheme 9

143

PRODRUGS OF VARIOUS FUNCTIONAL GROUPS

pound (66) with an aldehyde, usually formaldehyde, and a primary or secondary aliphatic amine (63). Aromatic amines do not usually undergo this reaction. Mannich base prodrugs are regenerated by chemical hydrolysis without enzymatic catalysis in the reverse direction of their formation.40 Transformation of an amide to an N-Mannich base introduces a readily ionizable amino functional group (67 Ð 68) that would allow the preparation of sufficiently stable derivatives with greatly enhanced water solubility at slightly acidic pH. Clinically useful N-Mannich base prodrugs include rolitetracycline and hetacillin. The highly water-soluble rolitetracycline (70) is an N-Mannich base of tetracycline (69) with pyrrolidine and is decomposed to tetracycline quantitatively with a halflife of 40 minutes at pH 7.4 and 35 C.41 Since the decomposition of N-Mannich bases does not rely on enzymatic catalysis, the rate of hydrolysis is the same in plasma and in buffer. Hetacillin (71) is an example of a cyclic N-Mannich basetype prodrug, which is formed by condensation of ampicillin with acetone. The prodrug is readily converted back to the active ampicillin and acetone, with a half-life of 15–20 minutes at pH 4–8 and 35 C.42,43 The advantage of hetacillin is its higher stability in concentrated aqueous solutions as compared to ampicillin, which has a more nucleophilic amine that would react with the strained b-lactam ring. OH

O

HO

OH O OH

H

H

O ACD/LogP

ACD/LogD pH 7 pH 1

–1.187±0.750

–4.78 –4.29

0.150±0.750

–3.02 –4.84

R1 = R2 = H

1.350±0.320

–1.54 –1.72

R1,R2 = >C(CH3)2

2.301±0.901

–1.61 –0.29

R N R=H H (69) Tetracycline OH (70) Rolitetracycline R = CH2 N

N

O (1) Ampicillin

H H R1

NH

S

N R2

(71) Hetacillin

N O

COOH

8.4.2.2. N-a-Acyloxyalkyl Derivatives N-a-Acyloxyalkylation has become a commonly used approach to obtain prodrug forms of various NH-acidic drug substances such as carboxamides, carbamates, ureas, and imides. This is because N-a-acyloxyalkyl derivatives (72) combine high in vitro stability with enzymatic lability. The derivatives are cascade or double prodrugs. The regeneration of the parent drug occurs via a two-step mechanism, the enzymatic cleavage of the ester group followed by spontaneous decomposition of the N-a-hydroxyalkyl intermediate (73) (Scheme 10). The usefulness of this approach depends on the stability of the N-a-acyloxyalkyl derivative 72, its susceptibility to esterase-catalyzed hydrolysis, and the rate of decomposition of the intermediate 73. N-a-acyloxyalkyl derivatives of imides and secondary amides, as well as ring structures containing such moieties, showed normal ester stability.44 To make such N-a-acyloxyalkyl derivatives useful as prodrugs, the a-hydroxyalkyl intermediate (73) formed after the enzyme-catalyzed hydrolysis must decompose quickly to release the original

144

PRODRUG APPROACHES TO DRUG DELIVERY

O

R′

O

O

R′

O

Enzymatic

N

O

Fast

R′′

Drug

N R′′

CO2H

OH

Drug

(72)

NH R′′

CO2H

Drug

(73)

(74)

Scheme 10

drug molecule (74). The rate of the chemical decomposition step was found to correlate with the pKa of the acidic NH group; a pKa of less than 10.5 is required for instantaneous decomposition of N-hydroxymethyl derivatives. However, N-a-acyloxyalkyl derivatives (75) of primary amides, and other primary amide-type structures such as carbamates and sulfonamides, are extremely unstable in aqueous solution and quickly undergo decomposition to the corresponding N-hydroxymethyl derivatives, which are stable. Such derivatives of simple primary amides decompose by an elimination-addition mechanism involving a reactive N-acylimine intermediate (76) (Scheme 11). For imides and secondary O

O

H2O

O

O

Elimination

Drug

N H

O

R

Drug R

(75)

CO2H

N (76)

Drug

N OH H (77) Stable

Enzymatic hydrolysis

Scheme 11

amides, their inability to form an N-acylimine is believed to contribute to the stability of their N-a-acyloxyalkyl derivatives (72). At pH 4 and 37 C, half-lives of hydrolysis of N-a-acyloxyalkyl derivatives (75) range from 1 to 90 minutes, whereas at pH 7.4 the half-lives of hydrolysis are < 1 minute.45 The resulting Nhydroxymethyl derivatives (77) are rather stable; the half-life for the decomposition of N-(hydroxymethyl) benzamide is 183 hours at pH 7.4 and 37 C.45 However, aldehydes other than formaldehyde can be used to from N-a-hydroxyalkyl derivatives that are more unstable than N-hydroxymethyl analogs. For example, the halflife for N-(a-hydroxybenzyl)benzamide is only 6.5 minutes at pH 7.4 and 37 C. The use of aldehydes other than formadehyde may further expand the applicability of this approach to simple amides with pKa above 11.45 8.4.3. Prodrugs of Amines The presence of an amino group can affect a drug’s physicochemical and biological properties in several ways. These include (1) intermolecular or intramolecular

145

PRODRUGS OF VARIOUS FUNCTIONAL GROUPS

aminolysis leading to reactive and/or potentially toxic substances, (2) solubility problems when the drug is present with another ionizable functionality such as COOH (zwitterionic nature under physiological pH, potentially limiting its dissolution rate and/or its passive permeability), and (3) terminal free amino acid groups providing recognition sites for proteolytic enzymes, such as aminopeptidase and trypsin, present in the gastrointestinal tract lumen, the brush border region, and the cytosol of the intestinal mucosa cells. For all these reasons, prodrug approaches have been advocated for improving in vivo behavior of active compounds containing amino groups. 8.4.3.1. Amides Because of the relatively high stability of amides in vivo, N-acylation of amines was formerly of limited use in prodrug design. Only a few examples of simple amide prodrugs are known that are sufficiently labile in vivo; these include the N-L-isoleucyl derivative of dopamine46 and the N-glycyl derivative midodrine.47,48 With the use of proteases as prodrug-converting enzymes, amines can be coupled to peptide carboxylates, resulting in amide bonds cleavable by proteases (e.g., 34). Midodrine (79) is a glycinamide prodrug, and the therapeutic effect of orally administered midodrine is due to the major metabolite desglymidodrine (78), an a-agonist formed by deglycination of midodrine. Midodrine is rapidly absorbed after oral administration. The plasma level of the prodrug peaks after about half an hour, and declines with a half-life of approximately 25 minutes, while the metabolite reaches peak blood concentrations about 1 to 2 hours after a dose of midodrine and has a half-life of about 3 to 4 hours. The absolute bioavailability of midodrine (measured as desglymidodrine) is 93% and is not affected by food. Approximately the same amount of desglymidodrine is formed after intravenous and oral administration of midodrine. Midodrine has been used successfully in the treatment of neurogenic orthostatic hypotension and, more recently, in the treatment of dialysis hypotension. It acts through vasoconstriction of the arterioles and the venous capacitance vessels, thereby increasing peripheral vascular resistance and augmenting venous return, respectively. The prodrug is a unique agent in the armamentarium against orthostatic hypotension since it has minimal cardiac and central nervous system (CNS) effects.47,48 O O

N OH H

R (78) Desglymidodrine R = H R = -COCH2NH2 (79) Midodrine

ACD/LogP 0.378±0.279 –0.319±0.595

ACD/LogD pH 7 pH 1 –1.14 –3.42 –0.83 –2.72

8.4.3.2. N-a-Acyloxyalkoxycarbonyl Derivatives Carbamates are of limited use in prodrug design due to their general resistance to enzymatic cleavage in vivo. The introduction of an enzymatically labile ester group in the carbamate structure could render them sensitive to esterase-catalyzed hydrolysis leading to activation. Thus, N-a-acyloxyalkoxycarbonyl derivatives (80) of primary and secondary

146

PRODRUG APPROACHES TO DRUG DELIVERY

O Drug

R′′

O

O Enzymatic

N

O

O

Drug

R′ O

R (80) R = H or alkyl

HO

N

R′′ O

OH

R R′

(81) R′′

Spontaneous

CHO

Drug NH

O

Spontaneous

Drug

R CO2

(83)

N

OH

R (82)

Scheme 12

amines may be readily transformed, as shown in Scheme 12, to the parent amine (83) in vivo.49,50 Esterase-catalyzed hydrolysis of the ester moiety in these derivatives leads to an unstable a-hydroxyalkoxycarbonyl intermediate (81) which spontaneously decomposes into the parent amine via a labile carbamic acid (82). These a-acyloxyalkyl carbamate derivatives are neutral and combine high stability in aqueous solution with high susceptibility to enzymatic reconversion to the active agent triggered by hydrolysis of the terminal ester functions; they may be promising reversible prodrugs for amino-containing compounds. These are used in the preparation of orally active aminomethyl-THF 1b-methylcarbapenems (56 ! 57). This approach has also been applied to peptides and peptidomimetics in order to improve their unfavorable physicochemical characteristics (e.g., size, charge, hydrogen-bonding potential), which prevent them from permeating biological barriers such as the intestinal mucosa, and by their lack of stability against enzymatic degradation.51,52 Many of the structural features of a peptide, such as the N-terminal amino group, the C-terminal carboxyl group, and the side chain carboxyl, amino, and hydroxyl groups, which bestow upon the molecule affinity and specificity for its pharmacological receptor, severely restrict its ability to permeate biological barriers and render it as a substrate of proteases. Bioreversible cyclization of the peptide backbone is one of the most promising new approaches in the development of peptide prodrugs. Cyclization of the peptide backbone enhances the extent of intramolecular hydrogen bonding and reduces the potential for intermolecular hydrogen bonding to aqueous solvent. Linking the N-terminal amino group to the C-terminal carboxyl group via an a-acyloxyalkoxy promoiety, as in (84), is an interesting approach that has been shown to work on a number of model peptides (Scheme 13). These cyclic prodrugs were designed to be susceptible to esterase-catalyzed hydrolysis (slow step), leading to a cascade of chemical reactions resulting in the generation of the linear peptide. In pH 7.4 buffer at 37 C, the cyclic prodrugs (84) were shown to degrade quantitatively to their corresponding linear peptides (87). In human plasma, the rates of hydrolysis of cyclic prodrugs were significantly faster

147

PRODRUGS OF VARIOUS FUNCTIONAL GROUPS

O

O

O Enzymatic

HN

O

HN

O

O

OH OH

Peptide

Peptide

(84)

O

(85) Spontaneous

HCHO

O HN

OH H2N

OH

Spontaneous

OH

Peptide Peptide

O

CO2

(87)

O

(86)

Scheme 13

than in buffer and were inhibited by paraoxon, a potent esterase inhibitor. In comparison to the linear peptides, the cyclic prodrugs were at least 70 times more permeable in cell culture models of the intestinal mucosa. 8.4.3.3. N-Mannich Bases N-Mannich bases have been used successfully to obtain prodrugs of amide- (see Section 8.4.2.1) as well as amine-containing drugs. Due to their rapid cleavage, with half-lives between 10 and 40 minutes at physiological pH and a pronounced decrease in their basicity of 3–4 pKa units,53 salicylamide N-Mannich bases (90) were evaluated as prodrug forms for primary and secondary amines (Scheme 14). In this case, the amide part of a Mannich base is the promoiety. To improve their stability in vitro and avoid stability-associated formulation problems, the hydroxyl group of the salicylamide N-Mannich bases (90) O

O N H

O

N

N H

Enzymatic

Drug

N Drug

O

O O

O (88)

R

HO

R

OH (89) Spontaneous HCHO

O Spontaneous

HN Drug O

NH2

(92)

+ HCHO

OH

(91)

Scheme 14

N H OH (90)

N Drug

148

PRODRUG APPROACHES TO DRUG DELIVERY

can be blocked by O-acyloxymethylation. O-Acyloxymethylated derivatives (88) were much more stable in acidic and neutral aqueous solutions than the parent salicylamide N-Mannich base (90) and could be readily converted to the latter in the presence of human plasma by enzymatic hydrolysis. In addition to providing an in vitro stabilizing effect, the concept of O-acyloxymethylation makes it possible to obtain prodrug derivatives of a given amine drug with varying physicochemical properties of importance for drug delivery, such as lipophilicity and water solubility. This can simply be effected by the selection of an appropriate a-acyloxymethyl group.54 8.4.3.4. Azo Prodrugs Amines have been incorporated into an azo linkage to form prodrugs that can be activated through azo reduction. In fact, sulfa drugs were discovered because of prontosil (93), an inactive azo dye that was converted in vivo to the active sulfanilamide (95) (Scheme 15). Clinically useful balsalazide (23), olsalazine (25), and sulfasalazine (26) are azo prodrugs of mesalazine (27). They are converted in vivo by bacterial azo reductases in the gut to the active 5-aminosalicylic acid (5-ASA or mesalazine, 27), which is responsible for their anti-inflammatory activity in the treatment of ulcerative colitis, as discussed earlier. H2N

NH2

S

O

O

O

O

NH2

H2N

[H]

NH2

S +

NH2

N N (93) Prontosil

(94)

NH2

H2N (95) Sulfanilamide

Scheme 15

8.4.3.5. Schiff Base Prodrugs Amines can form reversible Schiff bases with aldehydes and ketones. Although they are of limited use in small-molecule prodrugs, Schiff bases have been used to conjugate amine-containing drugs to polymers with carbonyl groups as macromolecular prodrugs for slow release and targeting. Doxorubicin was conjugated to polyethylene glycol (PEG) through a Schiff base linkage, and the resulting conjugate was found to release doxorubicin under the lysosomal acidic conditions in vitro and very slowly under physiological conditions. Moreover, the conjugate showed strong cytotoxic activity similar to that of free doxorubicin against lymphocytic leukemia cells in vitro.55 O

O

OH

OH OH OMe O

OH O

Me HO

O

N O (96)

O Me n

149

PRODRUGS OF VARIOUS FUNCTIONAL GROUPS

8.4.4. Prodrugs for Compounds Containing Carbonyl Groups 8.4.4.1. Schiff Bases and Oximes Schiff bases and oximes formed from ketones or aldehydes with amines or hydroxyl amines are chemically reversible under acidic or basic conditions. They could be used as prodrugs of compounds containing either an amine or carbonyl functionality. Oximes of enones (98, 100, and 101) have been used as prodrugs of contraceptive norethindrone (97) and levonorgestrel (99). The oximes are highly bioavailable and are converted in vivo through chemical hydrolysis to their corresponding active drugs.56,57 R′ OR H

H H

H

ACD/LogP (97) Norethindrone (98) Norethindrone-3-oxime (99) Levonorgestrel (100) Levonorgestrel-3-oxime (101) Norgestimate

R = H, R′ = Me, X = O R = H, R′ = Me, X = N-OH R = H, R′ = Et, X = O R = H, R′ = Et, X = N-OH R = Ac, R′ = Et, X = N-OH

3.384±0.348 3.866±0.607 3.916±0.348 4.398±0.607 5.002±0.611

X

A more recent application of oxime derivatives as prodrugs is the design of cascade prodrugs of dopamine agonists for the treatment of Parkinson’s disease. As shown in Scheme 16, enones such as S-(-)-6-(N,N-di-n-propylamino)-3,4,5,6,7,8hexahydro-2H-naphthalen-1-one (103) can be oxidized in vivo to catecholamines

N

In vivo hydrolysis

N

In vivo oxidation

N HO

OR

N (102) Oxime prodrug

O

OH (103)

(104) (–)-5,6-diOH-DPAT

Scheme 16

such as (
)-5,6-dihydroxy-2-(N,N-di-n-propylamino)tetralin ((
)-5,6-diOH-DPAT, 104) which are known as ‘‘mixed dopamine D1/D2 agonists’’ with potential utility in the treatment of Parkinson’s disease.58 Upon oral administration of catecholamines, the phenol and catechol moieties are rapidly metabolized to an extent that limits the therapeutic usefulness of these compounds. Enones such as 103 are prodrugs of such catecholamines and have been shown to improve their bioavailability and extend the duration of action. Compound 103 was found to be efficacious in vivo in models for Parkinson’s disease in the rat. To potentially further increase the usefulness of enone 103, a number of oxime ethers and oxime esters (102) were prepared as potential cascade prodrugs.59 It was found that the unsubstituted oxime and the acetyl-oxime induced a pronounced and long-lasting effect in vivo. The oxime derivatives were readily hydrolyzed under acidic and alkaline

150

PRODRUG APPROACHES TO DRUG DELIVERY

conditions. The fact that these oximes as well as 103 were inactive at the dopamine receptor, yet induced dopamine D1 and D2 receptor-related effects in vivo, suggested that they were acting as prodrugs and were being converted in vivo to the active species (104). Oximes can be acylated to make prodrugs, as in the above example and in the case of FLM 5011, which is a strongly lipophilic, poorly water-soluble, lipoxygenase inhibitor. The water solubility was mproved by using the succinate monoester prodrug. The bioavailability of FLM 5011 in rabbits after oral administration was markedly increased by its prodrug.60 OH ACD/LogD pH 7 pH 1

ACD/LogP N

O

R

(105) FLM 5011 (106) FLM 5011 succinate

R=H R = COCH2CH2COOH

7.70 5.23

7.705±0.553 7.708±0.599

7.70 7.71

8.4.4.2. Enol Esters Enol esters are rather stable, bioreversible derivatives of ketones and may be useful as prodrugs of agents containing enolizable carbonyl groups. As shown in Scheme 17, 60 -acetylpapaverine enol esters (107), prepared by acylation of the appropriate Li enolate with the respective anhydride, were hydrolyzed to 60 -acetylpapaverine (108) by esterases present in rat and human plasma, rat liver, and brain tissue supernatants. The intermediate 60 -acetylpapaverine cyclizes rapidly to coralyne (109), which has antitumor activity but has O

MeO MeO

N

O

MeO R

CH2

Esterases

NO

MeO

CH3

OMe

OMe OMe

OMe (107) 6′-Acetylpapaverine enol esters

(108) 6′-Acetylpapaverine MeO MeO

N

OMe OMe (109) Coralyne

Scheme 17

151

PRODRUGS OF VARIOUS FUNCTIONAL GROUPS

difficulty passing through the blood-brain barrier due to the presence of the positive charge. 60 -Acetylpapaverine enol esters are neutral and stable in aqueous solution and are potential prodrugs for enhancing delivery of coralyne to brain tissues.61 8.4.4.3. Oxazolidines The kinetics of hydrolysis of several oxazolidines derived from Tris and various aldehydes and ketones were investigated to explore their suitability as prodrug forms for b-aminoalcohols such as (
) ephedrine (110) and for carbonyl-containing substances.62-64 Oxazolidines were easily and completely hydrolyzed at pH 1–11 at 37 C. The hydrolysis rates were subject to general acid-base catalysis by buffer substances and depended strongly on pH. Most oxazolidines showed sigmoidal pH-rate profiles with maximum rates at pH 7–7.5. At pH 7.4 and 37 C, the half-lives of hydrolysis for the various ephedrine oxazolidines (111) ranged from 5 seconds to 30 minutes (Scheme 18). The reaction rates in neutral and basic solutions decreased with

R, R′ Me

Me

N Me O

R′

OH

N H

Me

R Oxazolidines (111)

(110) (–) ephedrine

H, H Et, H Ph, H 2-HOPh, H tBut, H Me, Me Cyclohexylidene

t1/2 (s) 5 18 300 5 1800 280 360

Scheme 18

increasing steric effects of the substituents derived from the carbonyl component and decreased with increasing basicity of oxazolidines. Oxazolidines are weaker bases (pKa 5.2–6.9) than the parent b-amino alcohol and are more lipophilic at physiological pH. Thus, oxalolidines can be considered as potentially useful prodrugs for drugs containing a b-amino alcohol moiety or carbonyl groups.62,63 Molecular complexation with cyclodextrins might be able to enhance the stability of oxazolidine prodrugs to make them potentially more useful.65,66 The stability characteristics of various N-acylated oxazolidines were also studied in an attempt to develop approaches which may solve the stability problems associated with the use of oxazolidines as prodrug forms. The N-acylated oxazolidines, including a carbamate derivative, were in fact found to be highly stable in an aqueous solution, but they also proved to be resistant to hydrolysis by plasma enzymes. The latter limits the use of N-acylated oxazolidines in prodrug design.64 8.4.4.4. Thiazolidines Thiazolidines of some a,b-unsaturated 3-ketone steroids including progesterone, testosterone, and hydrocortisone (112, 113) were prepared from the reaction with cysteine alkyl esters and cysteamines as potential prodrugs.67,68 The thiazolidines readily reverted to their parent steroidal ketones,

152

PRODRUG APPROACHES TO DRUG DELIVERY

thus meeting the requirements for a prodrug. Most of the thiazolidines were more lipophilic than their parent steroids, thereby imparting the desired change in the physicochemical properties to the derivatives of the steroids. Thus, they can function as bioreversible derivatives of the parent steroids, cysteines, and cysteamines. Cysteine derivatives are particularly attractive as promoieties due to the release of cysteine as the by-product upon activation. Both cysteines and cysteamines were also used as chemoprotective agents against side effects of chemotherapy and radiation therapy. Thus, thiazolidines of cysteines and cysteamines could be used as prodrugs for chemoprotection during chemotherapy and radiation therapy. O O HO

O OH

H H

S NH R

H (112) R = CO2Et (113) R = H

8.5. DRUG RELEASE AND ACTIVATION MECHANISMS Most prodrugs rely on enzymatic hydrolysis by esterases or proteases and, to a less extent, on chemical hydrolysis to achieve a one-step cleavage of the promoiety and the release of the original active drug (Figure 8.1Ai). These systems, as well as other one-step activation mechanisms (Figure 8.1Aii–iv) are simple and, in many cases, sufficient in achieving useful regeneration rates of the active agent. Otherwise, a cascade release/activation mechanism can be incorporated by taking advantage of autodegradation or intramolecular cyclization reactions to effect the release and activation of a prodrug. Some of the cascade strategies that have been employed in the design of prodrugs are shown in Figure 8.1B and will be briefly discussed here. 8.5.1. Cascade Release Facilitated by Linear Autodegradation Reactions A number of examples are known for the release of an active drug facilitated by a linear autodegradation process. This can be achieved through chemically unstable intermediates such as a-hydroxy amines, amides, and esters. Many of the double prodrugs discussed earlier belong to this category. Another interesting linear autodegradation process often used in prodrug design involves an electron ‘‘push and pull’’ mechanism through a conjugative aromatic ring that is linked to a good leaving group such as an ester in the para benzylic position. In such an approach, an electron-donating amino or hydroxy is masked as an electron-withdrawing nitro group or an amide or ester group in the prodrug.

153

DRUG RELEASE AND ACTIVATION MECHANISMS A. Simple one-step activation i. Hydrolysis

O Drug

X

Drug

OH

HX R

O R

O

Enzymatic

R

X

Drug

HX Drug

O R

ii. Oxidation

R

X = O, NH

Enzymatic

Drug

or Drug

OH

OH

O

Enzymatic [O]

Drug

e.g., nabumetone

OH

O iii. Reduction Enzymatic [H]

N Drug

R-Ar N

iv. Decarboxylation

O

Enzymatic

Drug

H2N Drug

e.g., sulfasalazine

Drug H

e.g., L-dopa

R-Ar NH2

OH

dopamine

CO2

B. Cascade release/activation mechanisms initiated by an enzymatic triggering step i. Linear releasing system

O R

R′ O

X

Drug

R′

Enzymatic

O R

EWG

HO

X

Drug

OH

R′

Enzymatic

HX Drug

O H

O

O

X = O, NH, O

,

N

O O S

,

N H

EDG

EDG

HX Drug X Drug

X Drug

O

O

EWG = NO2, N3, RCONH, RCO2 EDG = NH2, NHOH, OH

X=

O

O

N H

O

O P

O O S

O

ii. Cyclization releasing system

Masked Nu O X Drug

Nu O X Drug

O

Nu

+ HX Drug Masked nucleophiles NO2

N3

H N

R

O

O

O

R O

H N

R O

O

R

O O

iii. Cyclization to form cyclic drug

Masked Nu O X R′ Drug

Nu O X R′ Drug

Nu HX R′ or H2O

X R′

O

Drug

Nu or

X = O, NH

Drug X = CH2

Figure 8.1. Examples of drug-release/activation mechanisms.

Upon unmasking via reduction or hydrolysis, the resulting electron-donating group will be able to push electrons through the conjugative system to the para position, leading to the cleavage of the benzylic carbon-oxygen bond. The rate of this cleavage is not enzyme-dependent, but rather relies on the electron-pushing capability of the unmasked electron-donating group and the electron-pulling ability of the

154

PRODRUG APPROACHES TO DRUG DELIVERY

leaving group. The formation of the negatively charged species can serve as the activation mechanism of the drug. This approach has recently been used in our effects to develop a novel and superior class of nitroaryl phosphoramides as potential prodrugs for nitroreductase-mediated enzyme-prodrug therapy.33 Several nitroaryl phosphoramides were designed and synthesized, each with a strategically placed nitro group on the benzene ring in the para position to the benzylic carbon (Scheme 19). Compound 114 is a cyclophosphamide analog

NH O P O N(CH2CH2Cl)2

NTR

(115) NH NTR O P O N(CH CH Cl) 2 2 2 HO

(41) H2N O

P

(40)

(116)

NH O P O N(CH CH Cl) 2 2 2 N H

NTR

O

N(CH2CH2Cl)2

O HO N H

(117) H2N

O

HO

O2N

NH O P O N(CH2CH2Cl)2

NH O P O N(CH2CH2Cl)2

(114)

O2N

HO NH

HO NH

O2N

P

(118)

O

P O

HO N H

(119)

O

H2N

N(CH2CH2Cl)2

N H

NH O P N(CH2CH2Cl)2

(120)

N(CH2CH2Cl)2

(121)

116, 118, and 121 could cross-link DNA strands, leading to cytotoxicity.

Scheme 19

with the cyclophosphamide ring fused with a benzene ring and a nitro group placed in the para position to the benzylic carbon. Compound 41 is a 4-nitrophenyl substituted cyclophosphamide analog, and 40 is an acyclic nitrobenzyl phosphoramide mustard (LH7). The nitro group is a strong electron-withdrawing group (Hammett sp electronic parameter ¼ 0.78) and is converted to an electron-donating hydroxylamino group (sp ¼
0.34) upon nitroreductase reduction. This large difference in electronic effect (
sp ¼ 1.12) is exploited to effect the formation of the highly cytotoxic phosphoramide mustard or like reactive species. After reduction by nitroreductase (NTR), the resulting hydroxylamines 115, 117, and 119 relay their electrons to the para position and facilitate the cleavage of the benzylic C

O bond, producing the anionic cytotoxic species phosphoramide mustard 121 or like reactive species 116 and 118. Structurally, the phosphoramide portion in 116 and 118 closely resembles phosphoramide mustard 121, the reactive alkylating agent produced following the metabolic activation of cyclophosphamide in the liver, and could also be the ultimate cytotoxic alkylating agent. Phosphoramide mustard is the proven cytotoxic metabolite of cyclophosphamide, a successful clinical anticancer prodrug that requires cytochrome P450 activation in the liver. These nitroaryl phosphoramides in combination with nitroreductase could effectively move the site of activation from liver in the case

DRUG RELEASE AND ACTIVATION MECHANISMS

155

of cyclophosphamide into nitroreductase-expressing tumor tissues. All compounds were shown to be excellent substrates of E. coli nitroreductase, but with varying degrees of cytotoxicity against nitroreductase-expressing V79 and SKOV3 cells. Compounds cis-41 and trans-41, the best of the cyclic series, were over 22,000 more cytotoxic in nitroreductase-expressing Chinese hamster V79 cells and 40, the acyclic compound LH7, was 167,500 more cytotoxic in the same cell line, with an IC50 as low as 0.4 nM upon 72-hour drug exposure. This level of activity is about 100 more active and 27 more selective than CB1954. Even when the V79 cells were exposed to each test compound for 1 hour before the media were replaced with non-drug-containing fresh media, the IC50 was 10 nM, which was about 30 lower than that of CB1954 (31). The high selectivity of cis-41, trans-41, and 40 was reproduced in SKOV3 human ovarian carcinoma cells infected with an adenovirus expressing E. coli nitroreductase. Enzyme kinetic analysis indicates that compound 40 was a much better substrate of E. coli nitroreductase with a specificity constant 20 that of CB1954. 8.5.2. Cascade Release Facilitated by Intramolecular Cyclization Reactions Intramolecular reactions are usually thermodynamically favored over intermolecular reactions because they have lower activation energy and therefore more stable transition states. The lower activation energy is attributed to a better entropic situation. When a reaction is performed between two different reactants, the two molecules need to collide in a specific orientation and the reaction leads to a decrease in the number of molecules, resulting in an increase in order and therefore a loss of entropic energy. In an intramolecular reaction, the two reaction centers, e.g., a nucleophile and an electrophile, are both present within the same molecule and are in a good position to interact and form the cyclic product. The positioning of the two reaction centers within the same molecule is very important for the reaction to take place. Steric factors resulting in less flexible molecules and better positioning of the two reaction centers would lead to increased reaction rates. Generally speaking, intramolecular reactions leading to the formation of 5- or 6-membered rings are much more favorable and occur at faster rates. A series of alkylaminoalkyl carbamates of 4-hydroxyanisole (122) were evaluated as prodrugs of the melanocytotoxic phenol (123) that could be activated through intramolecular cyclization (Scheme 20).69 The carbamates were relatively stable at low pH but released 4-hydroxyanisole cleanly in a nonenzymatic fashion at pH 7.4 at rates that were structure-dependent. A detailed study of the N-methyl-N-[2-(methylamino)ethyl]carbamate showed that generation of the parent phenol followed first-order kinetics with t1=2 ¼ 36:3 minutes at pH 7.4, 37 C, and was accompanied by formation of N,N-dimethylimidazolidinone (124). In comparison, the related derivative with three methylene units between the two N atoms releases the phenol at a much slower rate with t1=2 ¼ 942 minutes. These basic carbamates are examples of cyclization-activated prodrugs in which generation of the active drug is not linked to enzymatic cleavage but rather depends solely upon a predictable, intramolecular cyclization-elimination reaction.

156

PRODRUG APPROACHES TO DRUG DELIVERY

R O

N

O (CH2)nNHR′

+

37 °C

O

MeO

OH

pH 7.5

N R′

(CH2)n

MeO

(122)

R N

(123) n 2 2 2 2 2 3 3

(124) R, R′

t1/2 (min)

Me, Me Et, Et Me, H H, Me H,H H,H Me,Me

36.3 118 304 335 724 910 942

Scheme 20

The terminal amino group in the above system could be masked as an amide, thus avoiding the stability problem encountered when using the basic carbamates as prodrugs. Unmasking of the amide could be catalyzed by a specific protease in vivo at certain target sites, thus achieving target specificity. Another alternative is to use o-nitroaromatic, as in compounds 125 (Scheme 21), which could be

FUDR O NO2

FUDR O NH2

R R

R R

[H] n

O R R

HN pH 7.4

n

n 37 °C

(125)

(126)

(127)

+ FUDR

(128)

n

R

t1/2 (min)

0 0 1 1

H Me H Me

14 ~0 4 2

Scheme 21

converted to a nucleophilic aromatic amine upon bioreduction in hypoxic tumor tissues or by other reductases delivered to targeted cells.70–72 Kinetic analysis of the cyclization activation process indicates that the addition of two a methyl groups to the ester carbonyl would restrict the rotational freedom of the ground state molecule and promote the cyclization reaction. The nitro group can be reduced in vivo to a nucleophilic aromatic amine with a low pKa (90% of ovarian and other gynecological cancers and at high to moderate levels in brain, lung, and breast carcinomas.51,54–56 Cancers of the endometrium, kidney, and colon have also been found to frequently express FR.50–59 Notably, the FR gene was recently mapped to region 11q13, a chromosomal locus that is amplified in >20% of tissue samples from breast and head/neck tumors.60,61 FR expression may also be dependent on the histological classification of a tissue or cancer. For example, using a recently developed quantitative assay for measuring functional FR in cells and tissues, it was found that normal ovarian tissue and the mucinous form of ovarian cancer express very low levels of FR (1 pmol FR/ mg protein; unpublished data, Endocyte, Inc., West Lafayette, IN). However, serous ovarian carcinomas express high amounts of FR, with the average expression level being 30 pmol FR/mg protein, or about 30-fold higher than that of the normal

176 TABLE 9.2

RECEPTOR-MEDIATED DRUG DELIVERY

FR Levels in Various Human Carcinoma Tissues IHC55

Tissue

% Positive

RT-PCR52 N

% Positive

Other Studies N

% Positive (N) 91% (34),62 90% (136),58 83% (40)63 64% (25)63

Ovarian

93%

56

100%

4

Endometrial Breast Lung Adenocarcinoma

91% 21% 33% n.d.

11 53 18

100% 80% 33% n.d.

3 5 3

Colorectal Kidney Prostate B-cell lymphoma Brain Mets. Head and neck Pancreas Bladder

22% 50% 0% 0% n.d. 80% n.d. 13% 0%

27 18 5 21

20% 100% n.d. n.d. 100% n.d. 50% n.d. n.d.

5 4

5 8 14

50% (49)64 Adenocarcinoma 90% (10)64 67% (3)63

8 4

n.d., not determined.

ovary. Endometrioid and metastatic ovarian carcinomas also express FR, although not as much as the serous form. Interestingly, others have observed a strong correlation between FR expression and both the histological grade and stage of the tumor.58 In general, highly dedifferentiated metastatic cancers express considerably more FR than their more localized, low-grade counterparts. 9.5.2. Expression of FRs in Normal Tissues FRs have also been detected in normal tissues, particularly those involved in the retention and uptake of the vitamin. For example, the choroid plexus in the brain expresses high levels of FR. However, the receptor is primarily localized to the brain side of the blood-brain barrier, where it is inaccessible to blood-borne folates or folate-drug conjugates.59,65,66 The FR has similarly been located on the apical membrane surface of the intestinal brush border,67 but again, access to these docking sites requires either escape into the lumen of the intestine or oral ingestion of the folates. FR is also expressed at high levels in the proximal tubules of the kidney, where it is believed to capture folates (and small molecular weight folate-drug conjugates) prior to their urinary excretion and then return them back into circulation via a transcellular reabsorption process.68–70 Recent reports further reveal FR expression on activated but not resting macrophages,71,72 an observation that could find utility in the treatment of autoimmune diseases. And finally, FR has also been

FOLATE-MEDIATED DRUG DELIVERY

177

detected at low levels in a few other normal tissues,52 but its relative expression level in these tissues is very low compared to that of many FR-positive cancers. This latter finding is evidenced by the fact that (1) the FR has been reliably employed as a tumor marker for many years,50–59 and, more importantly, (2) intravenously administered folates or folate-drug conjugates accumulate predominantly in FR-expressing tumor and kidney tissue.73–75 In summary, FR is expressed on the apical membrane surface of some normal polarized epithelia, where it is largely inaccessible to folates and their drug conjugates present in the blood. FR is also expressed on the surfaces of many malignant cells, where it is fully accessible to parenterally administered folate-drug conjugates due to the collapse of membrane polarization associated with transformation. Since 80% of human cancers arise from polarized epithelia, and since FR is further up-regulated in many of these malignancies, the FR has emerged as a useful target for receptor-directed therapies for cancer. The fact that FRs bind folate-drug conjugates with high affinity (10 9 M) and that the conjugates are subsequently transported nondestructively into the target cells only adds to the utility of this strategy for tumor-specific drug delivery. 9.5.3. Applications of Folate-Mediated Drug Delivery Initial studies on folate conjugate targeting were conducted with radiolabeled and fluorescent proteins (drug surrogates) covalently attached to folic acid.49 These conjugates were shown to bind and become internalized by FR-positive cells via a nondestructive, functionally active endocytic process.49 When FR was subsequently identified as a major tumor-associated antigen,50,76 much effort was quickly devoted to determining the types of attached cargo that might be easily targeted with folic acid. These studies have revealed that conjugates of radiopharmaceutical agents,73,74,77–82 magnetic resonance imaging (MRI) contrast agents,83 low molecular weight chemotherapeutic agents,17,84 antisense oligonucleotides and ribozymes,85–89 proteins and protein toxins,49,90–94 immunotherapeutic agents,95–98 liposomes with entrapped drugs,99–103 drug-loaded nanoparticles,104–106 and plasmids107–115 can all be selectively delivered to FR-expressing cancer cells. Indeed, the major limitation associated with the above targeting efforts appears to be the intrinsic permeability of the tumor. Thus, where perfusion barriers do not limit access to FR-expressing tumor cells, folate conjugate binding, FR-mediated endocytosis, and intracellular drug release are readily achievable if the fundamental principles outlined above are followed. In the remainder of this chapter, we shall illustrate the techniques of folatetargeted radiodiagnostic imaging, chemotherapy, and immunotherapy. However, the reader is encouraged to review the listed references if information on other folate conjugates is desired. 9.5.3.1. Tumor Targeting Through the FR: Radiodiagnostic Imaging The field of nuclear medicine has been revitalized with the advent of tissue-specific radiopharmaceutical targeting technologies. Ligands capable of concentrating at

178

RECEPTOR-MEDIATED DRUG DELIVERY

pathological sites have been derivatized with chelator-radionuclide complexes and then used as noninvasive probes for diagnostic imaging. Folate-targeted radiopharmaceuticals have been explored for the purpose of both (1) developing an imaging agent for the localization, sizing, and characterization of cancers and (2) obtaining ‘‘proof-of-principle’’ data regarding the ability of folic acid to deliver attached drugs to human tumors in vivo. Several animal models that contain tumors with FR levels similar to those found in common human carcinomas have been used to test uptake of folate-based radiopharmaceuticals in living organisms, including (1) nude mice with implanted human KB, MDA-231, or IGROV tumors, (2) C57BL/6 mice implanted with 24JK tumors, (3) Dupont’s c-neu Oncomouse,116 (4) DBA mice implanted with L1210A tumors, and (5) Balb/c mice implanted with syngeneic M109 tumors. This continually growing list of acceptable tumor models indicates that the location and nature of the tumor are relatively unimportant so long as the tumors express appreciable levels of FR. In 1999, Phase I/II clinical studies were initiated by Endocyte, Inc., to evaluate 111 In-DTPA-folate.75,82 Patients suspected of having ovarian cancer received a 5 mCi (2 mg) intravenous dose of the radiopharmaceutical, and whole body single photon emission computed tomographic (SPECT) images were taken 4 hours later to identify the location of the probe. Representative images from two enrolled patients are shown in Figure 9.4. The image displayed in Panel A shows that in a cancer-free patient, 111In-DTPA-folate (a folate-drug conjugate) primarily concentrates in the FR-positive kidneys, while the remaining tissues of the body effectively clear the radiopharmaceutical by 4 hours post injection. However, in addition to the kidneys (and to some extent the liver), the folate-targeted radiopharmaceutical accumulates in the widely disseminated malignant tissue in ovarian cancer patients (Panel B). Notably, a similar distribution pattern has also been demonstrated in patients receiving a new 99mTc-based radiopharmaceutical called EC20 (Ref. 81 and unpublished clinical observations). Taken together, and following the treatment of more than 45 enrolled subjects, we have noted a pattern of (1) consistent uptake of folate conjugates into malignant masses (including 1 cm metastatic abdominal lesions), (2) absence of uptake into benign masses, (3) consistent uptake by the kidneys, and (4) little or no uptake by other normal tissues. In summary, these clinical results have provided valuable ‘‘proof-of-principle’’ data confirming that folate-mediated tumor targeting also occurs in humans. It further suggests that folate-targeted radiodiagnostic imaging agents may be useful for noninvasively identifying the loci of pathological FR-positive tissue within patients. 9.5.3.2. Folate-Targeted Chemotherapy The fundamentals of folate-cytotoxin therapy were first illustrated by the targeted killing of FR-positive cells in vitro using folate conjugates of numerous protein synthesis–inhibiting enzymes.92 For example, folate conjugates of cell-impermeant, ribosome-inactivating proteins (e.g., momordin, saporin, gelonin, ricin A) were found to kill cultured FR-positive malignant cells without harming receptor-negative normal cells. The selectivity of this approach was confirmed by many important controls which demonstrated that (1) an excess of free folic acid quantitatively blocked folate-cytotoxin cell killing,

FOLATE-MEDIATED DRUG DELIVERY

179

Figure 9.4. Anterior SPECT images of two patients receiving 111In-DTPA-folate. (A) Image of a female patient without cancer. (B) Image of a female patient with stage IIIc ovarian carcinoma.

(2) the underivatized protein was not toxic to the target cells, and (3) pre-treatment of cells with phosphatidylinositol-specific phospholipase C, an enzyme that removes glycosylphosphatidylinositol-linked proteins (like the FR) from a cell’s surface, effectively blocked folate-cytotoxin cell killing. Importantly, the same folate-cytotoxin conjugates were also shown to selectively kill only malignant cells when cocultured in the same dish with ‘‘normal,’’ non-transformed cells.94 Overall, high activity (IC50 values of 1 nM) was observed using a number of FR-expressing cell lines. While it is tempting to speculate on the antitumor activity that may result from testing such folate-protein toxin conjugates in vivo, practical pharmaceutical considerations diminished the priority for their development. Instead, the focus shifted toward folate conjugates of conventional drug molecules. To date, both successes and failures with this approach have been published. In one study, researchers linked folate to a maytansinoid derivative (a natural product that blocks tubulin polymerization) via a disulfide bond. The conjugate was found to exhibit an IC50 of 50 pM for FR-positive KB cells but was nontoxic to FR-negative A375 cells,

180

RECEPTOR-MEDIATED DRUG DELIVERY

even though both cell lines were killed with equal potency (IC50 25 pM) by the free nontargeted DM1 maytansinoid.17 However, subsequent studies with ‘‘nonreleasable’’ folate conjugates of paclitaxel or a nitroheterocyclic bis(haloethyl)phosphoramidate prodrug were much less impressive.84,117 Unfortunately, none of the above drug conjugates was ever evaluated for activity against FR-positive tumors in vivo. Endocyte Inc., has recently collected data on the activities of a number of folatedrug conjugates both in vitro and in vivo. For example, folate has been conjugated to a potent small molecular weight DNA alkylator through a disulfide linker, and the resultant conjugate (referred to as EC72) was found to promote target cell killing in vitro (IC50  3 nM; unpublished data). Although the toxicity of the EC72 conjugate could be quantitatively blocked by the presence of excess free folic acid (to demonstrate FR specificity), neither EC72 nor the underivatized drug could effectively kill 100% of the cancer cells in vitro, possibly because of an intrinsic resistance to the mechanism of the drug’s activity. Nevertheless, despite this recognized limitation, the EC72 conjugate was evaluated in a pilot study using Balb/c mice bearing FR-positive M109 tumors. The study had two goals: (1) to determine if daily treatment with EC72 could prolong the lives of FR-positive tumor-bearing mice beyond that which the parent drug could do alone when tested under an identical dosing regimen and (2) to examine the pathological effects of EC72 treatment on normal tissues, including the FR-positive kidneys. As shown in Figure 9.5, all control mice died 22 days following an intraperitoneal inoculation with M109 cells, while a 39% increase in life span was observed

100

80

% Survival

39% ILS 60

40 178% ILS PBS

20

Parent drug EC72 0 0

10

20

30

40

50

60

Days post tumor implantation

Figure 9.5. Survival of treated M109 tumor-bearing mice. Four days post inoculation, Balb/c mice bearing intraperitoneal M109 tumors were treated once daily with either the unmodified parent drug or the folate-derivatized drug. ILS: increased life span.

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181

for the animals treated with the unmodified drug. More importantly, animals treated with the EC72 conjugate lived on average 178% longer. Following euthanasia, major organs were collected from both the EC72 and parent drug–treated animals, and they were sent to a certified pathologist for examination. The nontargeted drug-treated animals were found to suffer from massive myelosuppression (which is a characteristic dose-limiting side effect of the parent drug’s therapy), and all of the animals in this cohort died from obvious drug-related side effects. In dramatic contrast, animals treated with EC72 (folate-SS-drug) displayed no evidence of myelosuppression or kidney damage. Further, examination of blood collected from EC72-treated animals indicated normal blood urea nitrogen and creatinine levels following 30 consecutive daily injections with the conjugate. We concluded from these results that the use of folate-drug conjugates may be an effective form of chemotherapy that does not cause injury to normal tissues, including the FR-positive kidneys. While the latter conclusion was surprising, these observations do support the hypothesis that the FRs in the kidney proximal tubules function primarily to shuttle scavenged folates (or small folate-drug conjugates) back into systemic circulation rather than to deliver the conjugates into the kidney cells.69,70 9.5.3.3. Folate-Targeted Immunotherapy 9.5.3.3.1. Intracellular Delivery versus Cell-Surface Loading In contrast to most hormone receptors, not all FRs endocytose following ligand binding.118 Rather, a substantial fraction of the occupied FRs remain on the cell surface as a means of storing folates for use at a later date. Since folate-drug conjugates interact with FR in the same manner as free folate,49 it was of no surprise to learn that a fraction of these conjugates also remains extracellularly bound.49,93 This dual destiny of occupied FR obviously allows for two distinct uses of the folate-targeting technology: (1) delivery of folate-drug conjugates into FR-expressing cells and (2) decoration of FR-expressing cell surfaces with folate-drug conjugates. This latter application has led to the advent of folate-targeted immunotherapy,119 as outlined below. Based on the ability of folate to position an attached drug on the surface of an FR-expressing cancer cell, it was recognized that a malignant cell could be converted from its normal immunologically invisible state (the condition that permits its proliferation in vivo) to a state where it is vividly recognized as foreign by the immune system (and consequently subject to immune-mediated elimination) simply by targeting a highly immunogenic antigen to its cell surface. This concept is presented in Figure 9.6. Different from targeted chemotherapy, this treatment begins with a series of subcutaneous inoculations of a hapten-based vaccine to stimulate production of anti-hapten antibodies in the patient. After induction of an adequate antibody titer, a folate-hapten conjugate is administered to enable ‘‘marking’’ of all FR-expressing tumor cells with the hapten. This process rapidly promotes the tumor cell’s opsonization with the previously induced endogenous anti-hapten antibodies. Mechanistically, the folate-hapten conjugate forms a molecular ‘‘bridge’’ between the tumor cell and the endogenous circulating anti-hapten

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Figure 9.6. Folate-targeted immunotherapy strategy.

antibody. Ultimately, this marking process enables Fc receptor-bearing effector cells, such as natural killer (NK) cells and macrophages, to recognize and destroy the antibody-coated tumor cells. Although we have found this folate-targeted immunotherapy to be effective in tumor-bearing rodent models, we have also noted that complete and reproducible cures are obtained only when the animals are concomitantly dosed with low levels of the cytokines, interleukin-2 and interferon-a.119 The purpose of adding these cytokines is to stimulate those immune effector cells (like NK and macrophage cells) that are responsible for killing opsonized tumor cells. Importantly, the low cytokine dose levels applied in this technique were found not to produce effective antitumor responses in the absence of the folate-hapten conjugate. Although the exact effector mechanisms involved in this therapy have not yet been established, there is strong evidence to suggest that the cured animals develop independent cellular immunity against the tumor cells and that this cellular immunity prevents the animals from growing tumors when rechallenged with the same malignant cells, even without additional treatment.119 These findings also support the notion that a long-term, perhaps T cell-specific immunity, develops during the hapten-mediated immunotherapy, and that a shift of the host’s antitumor response from humoral to cellular immunity occurs. Because this therapy has generated cures in multiple tumor-bearing animal models, and since the complete therapeutic regimen (vaccination and drug therapy at dose levels approaching 100-fold of the human dose equivalent) did not produce test article–related toxicities in toxicology studies, the strategy was approved for clinical trial by the FDA for treatment of advanced

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ovarian and renal cell cancers beginning in 2003. Results of these clinical studies should provide much information on the strengths and weaknesses of folatetargeted cancer therapies in humans.

9.6. CONCLUSIONS Nature has designed biological membranes to serve as formidable barriers against the unwanted entry of harmful agents into cells. Life, however, cannot exist without the cell’s ability to selectively capture and internalize certain biosupportive molecules. Receptor-mediated endocytosis is but one mechanism by which cells retrieve required molecules from their environment, and it is also a powerful means for delivering normally impermeable molecules into target cells for medicinal purposes. The list of available ligands for use in this exploitive technique keeps growing, as does the potential for inventing alternative drug delivery strategies. It is important, however, to remember that not all ligand-receptor systems will function in the same manner. Many variations exist among cell types, including their level of receptor expression, internalization rate, ligand affinity, intracellular compartmentalization, recycling capabilities, etc. Thus, in spite of recent technological advances, many challenges remain for the future development of receptor-targeted therapies. The most significant issues will undoubtedly be related to the confirmation of target tissue specificity (i.e., receptor distribution among target and nontarget tissues) as well as the design of releasable linkers for certain ligand-linker-drug conjugates. Receptor-mediated delivery technology is undoubtedly influencing rational drug design, and it is our hope that the scientific and pharmaceutical communities will continue to invest in this exiting new field of medicine.

ACKNOWLEDGMENT This work was supported in part by grants from Endocyte, Inc., and the NIH (CA89581).

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10 ORAL PROTEIN AND PEPTIDE DRUG DELIVERY RICK SOLTERO Pharma Directions, Inc.

10.1. Introduction 10.2. Overcoming physiological barriers with formulation approaches 10.2.1. Enzyme inhibitors 10.2.2. Absorption enhancers 10.2.2.1. Nanoparticles 10.2.2.2. Emulsions 10.2.2.3. Micelle formulations 10.2.2.4. Liposomes 10.2.3. Chemical modifications 10.3. Improving the oral activity of calcitonin and insulin through chemical modification 10.4. Conclusions References

10.1. INTRODUCTION Endogenous peptides play a pivotal role in almost all regulatory processes of the body functions and act with high specificity and potency. Their vast therapeutic potential has prompted biomedical scientists to greatly expand research into the identities of biologically active polypeptides and proteins and their activities.

Drug Delivery: Principles and Applications Edited by Binghe Wang, Teruna Siahaan, and Richard Soltero ISBN 0-471-47489-4 # 2005 John Wiley & Sons, Inc. 189

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Concurrently, pharmaceutical companies around the world have endeavored to develop processes for producing therapeutically active entities at commercial scales. As a result of this research, the availability of biotechnology products to develop is no longer an issue. At the present time, over 350 peptide and protein drugs are in various stages of clinical development. Many other candidates will likely be identified as genomic studies successfully translate genetic data into knowledge about proteins and their functions. The broad availability of structurally diverse peptides possessing a spectrum of pharmacological effects has not been matched, however, by their clinical development, mostly because of the poor delivery properties of peptide- and protein-based products. Today, the most common means for administering these protein drugs remains injection (i.e., intravenous, intramuscular, or subcutaneous administration). Patient compliance with drug administration regimens by any of these parenteral routes, is generally poor and severely restricts the therapeutic value of the drug, particularly for diseases such as diabetes. Delivering therapeutically active proteins and peptides by any route other than the invasive injection methods has been a challenge and a goal for many decades.1 Among the alternate routes that have been tried with varying degrees of success are the oral, buccal,2 intranasal,3 pulmonary,4 transdermal,5 ocular,6 and rectal7 approaches. The many benefits of oral administration of therapeutic agents make oral polypeptide delivery stand out from among these drug delivery alternatives. In addition to high levels of patient acceptance and long-term compliance, oral delivery also benefits the patient through avoidance of the pain and discomfort associated with injections; greater convenience; implementation of a more socially compatible dosing regimen; and the elimination of concerns about needle-related infections. In addition, a growing body of data suggests that for certain polypeptides, such as insulin, the oral delivery route is more physiological.8,9 Thus, oral delivery would be an ideal route if appropriate oral dosage forms of therapeutic peptides and proteins were available. The oral route of administration of these therapeutic agents, however, is among the most problematic delivery regimens. Drug delivery via the gastrointestinal (GI) tract requires relatively lengthy exposure to a multifaceted system that is designed to degrade nutrients and dietary materials into small molecules and to prevent the indiscriminate passage of macromolecules, as well as other large entities such as microbes or foreign antigens that may present dangers to the host. The various barriers, including physiological, chemical, and biochemical ones, for oral drug delivery are discussed in detail in Chapter 2 and therefore will not be presented in this chapter. In spite of the obstacles to oral delivery, substantial evidence suggests that pharmaceutical polypeptides are absorbed through the intestinal mucosa, although in minute amounts.10 Small amounts of polypeptide drugs can be absorbed by the action of specific peptide transporters in the intestinal mucosa cells.11 This suggests that properly formulated proteins or peptide drugs may be administered by the oral route with retention of sufficient biological activity for their therapeutic use.

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Designing and formulating a polypeptide drug for delivery through the GI tract requires a multitude of strategies.12,13 The dosage form must initially stabilize the drug while making it easy to take orally.14 It must then protect the polypeptide from the extreme acidity and action of pepsin in the stomach.15 When the drug reaches the intestine, the formulation must incorporate some means for limiting drug degradation by the plethora of enzymes that are present in the intestinal lumen.16 In addition, the polypeptide and/or its formulations must facilitate both aqueous solubility at near-neutral pH and lipid layer penetration in order for the protein to traverse the intestinal membrane and then the basal membrane for entry into the bloodstream.17 To accomplish this, formulation excipients that promote absorption may be required.18,19 Finally, when the modified polypeptide enters the systemic circulation, the structural modifications may add to the functionality of the drug, e.g., by extending its half-life in the circulation. However, any structural changes that may have been employed to enhance oral bioavailability must not interfere with receptor binding and uptake at the site of biological activity. In this chapter, we summarize the general approaches that have been used to successfully achieve the formulation goals for oral delivery: minimize enzymatic degradation; enhance intestinal absorption; maximize blood level reproducibility; deliver drug through the gut wall; and produce a palatable and acceptable dosage form. Then insulin will be used as an example to show how oral bioavailability has been achieved through chemical modification.

10.2. OVERCOMING PHYSIOLOGICAL BARRIERS WITH FORMULATION APPROACHES Oral delivery of polypeptides has been an ongoing challenge. Nearly every oral dosage form used for delivery of conventional small-molecule drugs has been used to explore oral delivery of polypeptides. Except for cases where the polypeptide has been chemically modified or where a proprietary absorption enhancer has been used, the results have been disappointing. There have been only a handful of human clinical trials where there has been adequate bioavailability and pharmacokinetics to suggest that commercializing an orally delivered polypeptide is feasible. Some of the approaches used to date are described in general terms in this section. For more in-depth discussions, readers should consult the numerous reviews that cover specific areas of research.20–26 10.2.1. Enzyme Inhibitors Researchers have evaluated the use of protease inhibitors with the aim of slowing the rate of degradation of proteins and peptides in the GI tract. The hypothesis was that a slow rate of degradation would increase the amount of protein and peptide drug available for absorption. For example, enzyme degradation of insulin is known to be mediated by the serine proteases trypsin, a-chymotrypsin, and thiol metalloproteinase insulin-degrading

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enzymes. The stability of insulin has been evaluated in the presence of excipients that inhibit these enzymes. Representative inhibitors of trypsin and a-chymotrypsin include pancreatic inhibitor and soybean trypsin inhibitor,27 FK-448,28 camostat mesylate,29 and aprotinin.30 Inhibitors of insulin-degrading enzyme include 1,10 phenanthroline, p-choromeribenzoate,31 and bacitracin.32 Ziv et al.33 reported the use of a combination of an enhancer, sodium cholate, and a protease inhibitor to achieve a 10% increase in rat intestinal insulin absorption. Another approach to enzyme inhibition is to manipulate the pH to inactivate local digestive enzymes. Lee et al. conducted studies demonstrating that oral absorption properties of salmon calcitonin can be modulated by changing intestinal pH. Reducing the intestinal pH in the GI tract, increased absorption of the intact peptide.34 A sufficient amount of a pH-lowering buffer that lowers local intestinal pH to values below 4.5 can deactivate trypsin, chymotrypsin, and elastase. 10.2.2. Absorption Enhancers Permeation enhancers improve the absorption of protein and peptides by increasing their paracellular and transcellular transports. An increase in paracellular transport is mediated by modulating the tight junctions of the cells, and an increase in transcellular transport is associated with an increase in the fluidity of the cell membrane. Chapter 2 describes in depth the various paracellular and transcellular transport pathways, which will not be discussed here. Paracellular permeation enhancers include calcium chelators, bile salts, and fatty acids. Calcium chelators, such as ethylendiaminetetraacetic acid (EDTA),5 act by inducing calcium depletion, thereby creating global changes in the cells, including disruption of actin filaments, disruption of adherent junctions, and diminished cell adhesion.35 Zonula occludens toxin (ZOT) acts specifically on the actin filaments of tight junctions,36 and chitosan triggers opening of the tight junction between the cells.37 Transcellular permeation enhancers include surfactants, medium chain fatty acids, nonionic surfactants,38 sodium cholate and other bile salts,39 and many other surfactants. 10.2.2.1. Nanoparticles Nanoparticles have been studied in recent decades as particulate carriers to deliver the protein and peptides drugs orally. This approach is supported by the literature, which states that particles in the nanosize range are absorbed intact by the intestinal epithelium, especially Peyer’s patches, and travel to sites such as the liver, the spleen, and other tissues.40–42 The proteins and peptides encapsulated in the nanoparticles are less sensitive to enzyme degradation through their association with polymers. The extended release of protein and peptide drug from the particles could have pharmacological and clinical significance. Many researches have demonstrated that protein and peptide encapsulated in the nanoparticles have better absorption through the GI tract compared to their native counterpart.43 The factors affecting uptake include the particle size of the

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particulate, the surface charge of the particles, the influence of surface ligands, and the dynamic nature of particle interactions in the gut. In one example, the interaction of nanoparticles consisting of hydrophobic polystyrene, bioadhesive chitosan, and (PLA-PEG) with two human intestinal cell lines was investigated and compared to the in vivo uptake in rats. After intraduodenal administration of chitosan nanoparticles in rats, particles were detected in both epithelial cells and Peyer’s patches. Chitosan nanoparticle seemed to be taken up and transported by adsorptive transcytosis, while polystyrene nanoparticles uptake was probably mediated by nonadsorptive transcytosis.44 In another example, insulin was encapsulated in nanosphere formulations using phase inversion nanoencapsulation. The encapsulated insulin was released from the nanospheres over a period of approximately 6 hours, was shown to be active orally, and had 11.4% of the efficacy of intraperitoneally delivered insulin.45 One problem in using nanoparticles for peptide and protein delivery is the erratic nature of nanoparticle absorption. For example, the proportion of intact particles reaching the systemic circulation was estimated to be generally below 5%.46 Considering the generally low encapsulation efficiency of the protein in the particulates, the overall oral bioavailability of proteins and peptides is not significant. 10.2.2.2. Emulsions Liquid emulsions have been used to deliver proteins and peptide orally. Emulsions are thought to protect the drug from chemical and enzymatic breakdown in the intestinal lumen. Drug absorption enhancement is dependent on the type of emulsifying agent, particle size of the dispersed phase, pH, solubility of drug, type of lipid phase used, etc. Water-in-oil microemulsions have been shown to enhance oral bioavailability of proteins and peptides.47,48 The lipid phase of microemulsions composed of medium chain fatty acid triglycerides increased the bioavailability of muramyl dipeptide analog.49 Many successful formulations are based on emulsion techniques. In these formulations, oil and water are mixed in such a way that small, uniformly shaped oil droplets are dispersed in the water phase (oil in water) or water droplets are dispersed in a continuous oil phase (water in oil). An emulsion appears as a cloudy suspension. When an oil-in-water emulsion has oil droplets so small as to produce a clear solution, then formulation is called a ‘‘microemulsion.’’ The oil phase of the emulsion can provide lipophilic proteins with some protection from enzymatic digestion while the product is in the intestinal tract. Water-in-oil microemulsion formulations have been developed for oral insulin delivery.50 10.2.2.3. Micelle Formulations A micelle system can be either water-based or oil-based. The use of a micelle formulation for poorly water-soluble drugs for systemic delivery has been well recognized. In recent years, the effective development of self-emulsifying microemulsions or mixed micelle-based lipid formulations products, such as Sandimmun Neoral (cyclosporin), Norvir (ritonavir), and Fortovase (saquinavir), has substantially increased interest in the application of lipid-based micelle formulation to improve oral delivery of poorly water-soluble drugs as well as protein and peptide drugs.51

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P. Modi patented a mixed micellar for administering insulin to the buccal mucosa using a metered dose inhaler.52 The formulation includes a micellar proteinic pharmaceutical agent, lauryl sulphate, salicylate, edetate, and at least one absorption-enhancing compound such as lecithin, hyaluronic acid, glycolic acid, oleic acid, linolenic acid, or glycerin. 10.2.2.4. Liposomes Liposomes have also been studied as a way to deliver peptides and proteins orally. Chapter 19 discusses in detail the use of liposomes as drug delivery vehicles. Therefore, only issues relevant to peptide and protein drug delivery are discussed here. Liposomes are prone to the combined degrading effects of the acidic pH of the stomach, bile salts, and pancreatic lipase upon oral administration. Compared to the parenteral route, there have been fewer attempts to develop oral formulations to deliver the protein and peptides using a liposome system. In cases where the encapsulated agents within liposomes have increased bioavailability, it is not clear whether the liposome was absorbed intact or if the lipid caused permeation of the released agent at the site of absorption.53 There are several reports on the intact liposomes uptake by cells in in vitro and in situ experiments;54–56 the results are, however, not convincing for the oral delivery of proteins with a liposome system. Attempts have been made to improve the stability of liposomes either by incorporating polymers at the liposome surface57,58 or by using GI-resistant lipids.59 10.2.3. Chemical Modifications Chemically modified proteins and peptides offer some significant advantages over native proteins. Proteins are inherently unstable to digestive enzymes; however, chemical modifications can be made that inhibit enzyme attack. Such modifications include prodrug approaches60 and permanent modifications.61 Prodrugs, including peptide prodrug derivatization, are covered in Chapter 8 in detail and therefore will not be discussed in this chapter. Permanent modifications, if conducted in the appropriate functional group, may increase the stability and oral bioavailability of a peptide or protein without compromising its biological activity. Various permanent modification approaches have been studied, especially with proteins. These include glycosylation (reference), pegylation (reference), cross-linking (reference), and other polymer conjugation. So far, two of the most successful examples in protein drug delivery using chemical modification are calcitonin and insulin.

10.3. IMPROVING THE ORAL ACTIVITY OF CALCITONIN AND INSULIN THROUGH CHEMICAL MODIFICATION Calcitonin is a 32-amino acid peptide hormone that participates in calcium and phosphorus metabolism. In mammals, the major source of calcitonin is from the parafollicular or C cells in the thyroid gland. Calcitonin contains a single disulfide bond, which causes the amino terminus to assume the shape of a ring. It is used to

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treat Paget’s disease of bone. It also may be used to prevent continuing bone loss in women with postmenopausal osteoporosis and to treat hypercalcemia (too much calcium in the blood). Chemical modification such as adding polyethylene glycol polymers can improve the in vivo characteristics of peptides. An example is CT-025, which is a diconjugate of recombinant salmon calcitonin (sCT).62 The chemical modification of sCT results in an orally available diconjugate that retains hormonal activity while having increased amphiphilicity. Modification can occur at sites where an amine is available. On sCT, there are sites at Cys-1, Lys-11, and Lys 18, as shown in Figure 10.1. Insulin is the major hormonal regulator of glucose metabolism. It is a 51-amino acid polypeptide that is produced and secreted by the b-cells of pancreatic islets.64,65 All known vertebrate insulins are composed of two polypeptide chains that are linked to one another by two disulfide bonds, one between CysA7 and CysB7 and the other between CysA20 and CysB19. A third, intrachain disulfide bond connects CysA6 and A11. The amino acid sequence among vertebrate species is also highly conserved. One-third of the amino acid residues in the two insulin chains are strictly invariant, and more than one-third of the remaining amino acids are either invariant or highly conserved, suggesting unique structural requirements O O

HN

Gly

nOCH3

7 Leu

Lys

Ser Gln

Leu

Cys 1

Val

Glu

Cys

Lys 11

Cys

V8 cleavage site

Leu

Thr Ser

His

Ser Asn

O

Lys 18

Leu

Lys

N H

Leu Pro

Tyr

Thr

Gln

Gly

Ser

Gly

Arg

Thr

Asn Thr

Thr

Pro

Amide

Figure 10.1. Chemical modification sites on sCT.63

O 7

nOCH3

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ORAL PROTEIN AND PEPTIDE DRUG DELIVERY

for receptor recognition and biological activity. Moreover, there is evidence that receptor binding is associated with a structural change that must be accommodated by insulin analogs if biological activity is to be preserved.66 For over 70 years, approved insulin products have been given to diabetic patients exclusively by parenteral administration. Due to the challenges and obstacles associated with oral administration of peptide drugs, no oral dosage form for insulin has been marketed to date.67 In general, studies of insulin’s oral bioavailability have shown uptakes that range from near zero to a small percentage of the administered dose.68–75 As part of continuing efforts to design and develop oral delivery dosage forms for peptide drugs, researchers have extensively investigated the effects of structural manipulations and formulation changes on the oral availability of insulin, These efforts have resulted in the preparation of libraries of amphiphilic oligomers that, when conjugated in certain patterns and number to the drugs, may be used to enhance their pharmacological performance. Moreover, when the drug-oligomer conjugates are used in combination with common formulation strategies, oral bioavailability can be dramatically increased, especially relative to that of the unconjugated, native drug. In previous reports,76–78 researchers showed that conjugation of properly designed amphiphilic oligomers to insulin provides insulin analogs that maintain solubility in aqueous and lipophillic matrices, are resistant to enzymatic degradation, have improved physicochemical properties for absorption across intestinal mucosa, and are orally active. These results also suggested that a particular insulin modification, oligomer-conjugation at the epsilon-amino group of lysine-29 on the B chain of insulin, would be a suitable oral insulin candidate.78 It has long been recognized that chemical modifications of proteins can improve bioavailability, delivery to systemic targets, and other pharmaceutical and pharmacological performance characteristics.79 Of the functional groups available for modification, the primary amino groups present at the N termini of the protein chain(s) and the e-amino groups of lysine residues are frequently selected, since most polypeptides and proteins have at least one of these sites and a wide variety of chemical agents are available that exhibit specificity for these functional groups. Insulin, for example, has three primary amino groups that are available for modification: the glycine and phenylalanine residues at the N termini of the A and B chains (GlyA1 and PheB1, respectively) and the e-amino group of lysine-29 on the B chain (LysB29). Several groups have reported that modification of GlyA1 causes diminished receptor binding and a loss of biological activity.7 Conversely, (MPEG) modification of PheB1 reduces the antigenicity of the protein, and PheB1 modification with glucosyl-PEG has been reported to suppress the association of insulin monomers/dimers into hexamers.80,81 Likewise, modification of either PheB1 or LysB29 has been shown to improve the pharmaceutical performance (e.g., increased plasma half-life, reduced immunogenicity and antigenicity, improved resistance to proteolysis, increased aqueous/ organic solubilities) of the insulin conjugates thus produced while not significantly

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compromising receptor binding or biological activity.82,83 A review by Brange and Volund provides detailed physicochemical and pharmacological characterization of numerous insulin analogs that have been developed over the past 70 years or are being investigated today.84 Fortunately, the pKa values of these three amino groups (300 days), whereas all control mice were dead by day 111. A clinical trial was also conducted in patients with advanced colorectal carcinoma of the lower intestinal tract.9,106 A dose of 20,000 enzyme units/m2 mAb-CPG2 conjugate was administered in the first step. This conjugate dose produced tumor CPG2 levels comparable to those found to be optimum in nude mice bearing xenografts. This treatment was followed 24–48 hours later by administration of a clearing agent, anti-CPG2 galactosylated Ab (220 mg/m2), to help remove mAb-CPG2 conjugates that were not bound to the antigen. In the last step, prodrug CMDA was injected over 1–5 days up to a total dose of 1.2–10 g/m2.

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Oral cyclosporine was coadministered to suppress the host immune response. From eight evaluable patients, there were four partial responses and one mixed response. A more recent clinical trial involving patients with colorectal carcinoma expressing CEA using a similar approach demonstrates that the median tumor: plasma ratio of enzyme (CPG2) exceeded 10,000:1 at the time of prodrug administration. Enzyme concentrations in the tumor were sufficient to generate cytotoxic levels of the active drug.107 A tumor response to the ADEPT was observed.107 Thus the results obtained are encouraging and prove the feasibility of the ADEPT at the clinical level. 11.3.1.2. ADEPT with b-Lactamase Activation of b-lactam-based prodrugs is based on the well-established b-lactam chemistry. It was demonstrated that a molecule attached to the 30 position of cephalosphorins was eliminated through a 1,4fragmentation reaction.11 Scheme 27 shows a general scheme of this reaction, H N

R O

O β-lactamase

N

X

O

H N

R

S β-lactamase

S N

O– O β-lactamase

Drug

O

OH

Drug OH

62a

62 (Prodrugs) H N

R O

O β-lactamase

S N O 62b

+

Drug

OH 62c (Activated drugs)

Scheme 27. Bioactivation of prodrugs by b-lactamase.

and Figure 11.11 shows the structures of the prodrugs of a vinca derivative (63), phenylenediamine mustard (64), doxorubicin (65), melphalan (66), paclitaxel (67), and mitomycin (68) prepared based on this chemistry.11 The first report of in vivo activity in a mAb-lactamase system used the b-lactamase from E. cloacae and a cephalosphorin-vinca alkaloid prodrug LY266070 (63, Figure 11.11).108 Nude mice implanted with human colorectal carcinoma were used for the investigation. The tumor inhibitory effects of the ADEPT with mAb-lactamase conjugate and LY266070 were superior to those produced by prodrug alone. The effects were also superior to those obtained when the vinca alkaloid (Figure 11.11) was attached directly to the mAb. Long-term regression of established tumors was observed in several dosing regimens, even in animals having tumors as large as 700 mm3 at the initiation of therapy.108

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ANTIBODY- AND GENE-DIRECTED ENZYME PRODRUG THERAPY

HO N CO2Me NH MeO

OH OH

S

O

O

O

N

Cl

S

O

N

HN

N

O

H N

HO

N

O

Me

H N

S

Cl

N

N

O

O O

NH

O

OH

O O

O

OH

64

63 O

O

OH

Cl

OH

N

Cl

OH OMe O H N

Me

S N

O

OH

HO

O

O

H N

O

N

O O

HOOC

S

NH

O

O O

OH

NH

O O

O

O

OH

66

65 O

O

S

C6H5

S N

O

H N

O

O

O

O

O

O

O O

OH

OH

NH O

O

O H N

O

Me

C6H5

O

O Me

C6HO 5

O

67 H N O

S N

H N

O

OH Me

OCONH2

O

OMe

O N

NH

O

68

Figure 11.11. Structures of representative prodrugs that are activated by b-lactamase.

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In a related study, Kerr et al. reported that ADEPT treatment with a combination of a mAb-lactamase conjugate and a prodrug of cephalosphorin-phenylenediamine mustard (64) in nude mice bearing subcutaneous 3677 human tumor xenografts produced regression in all the treated mice at doses that caused no apparent toxicity.109 At day 120 post tumor implant, four of five mice who had received this treatment remained tumor free. Significant antitumor effects were even seen in mice that had large (800 mm3) tumors before the first prodrug treatment. 11.3.2. GDEPT Unlike ADEPT, the major problem with GDEPT is delivery of the gene to the tumor—the same problem associated with cancer gene therapy. Almost all clinical trials currently being performed for cancer gene therapy rely on direct intratumor injection of the vector. This is not only a major limitation for uniform distribution of the delivered gene throughout the primary tumor, but it also fails to address the problem of delivery to sites of metastatic spread, which may be too numerous, invisible, or inaccessible for direct injection. Efforts to overcome this problem include engineering adenoviral vectors to alter their tropism for infection,110 use of cationic liposomes with surface ligands that are specific for tumors,111 and use of live attenuated Salmonella that can preferentially accumulate in tumors by an as yet unknown mechanism.112,113 Discussion of these efforts is beyond the scope of this chapter. A closely related approach to GDEPT is virus-directed enzyme prodrug therapy (VDEPT).114 The only difference between these approaches is that GDEPT involves both viral and nonviral vectors. For a more extensive discussion of GDEPT, readers are referred to earlier reviews.6–8 In this chapter, GDEPT will be introduced through two representative examples. 11.3.2.1. Hypoxia-Selective GDEPT A very promising approach to deliver the gene to solid tumors was recently described by Liu and colleagues.115 This approach takes advantage of the hypoxic/necrotic condition of solid tumors. The bacterial genus Clostridium comprises a large and heterogeneous group of grampositive, spore-forming bacteria that become vegetative and grow only in the absence (or low levels) of oxygen. Therefore, strains of these bacteria have been suggested as tools to selectively deliver the gene vector to the hypoxic and necrotic region of solid tumors.116–118 Liu and coworkers described, for the first time, the successful transformation of C. sporogenes, a clostridial strain, with the Escherichia coli cytosine deaminase (CD) gene.115 They showed that intravenous injection of spores of C. sporogenes containing an expression plasmid for E. coli cytosine deaminase into tumor-bearing mice produced tumor-specific expression of cytosine deaminase protein, an enzyme that converts the nontoxic prodrug 5-fluorocytosine (5-FC) (69) to the anticancer drug 5-fluorouracil (5-FU) (25d, Scheme 28). More importantly, significant antitumor efficacy of systemically injected 5-FC was observed following IV injection of these recombinant spores. The antitumor efficacy of the prodrug 5-FC following a single IV injection of the recombinant

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ANTIBODY- AND GENE-DIRECTED ENZYME PRODRUG THERAPY

O

NH2 F

N O

N H

O

H

69 5-Fluorocytosine (5-FC)

F

HN

Cytosine deaminase

N H

H

25d 5-Fluorouracil (5-FU)

Scheme 28. Bioactivation of 5-FC by cytosine deaminase.

spores is equivalent to or greater than that produced by the maximum tolerated dose of the active drug 5-FU given by the same schedule. A major advantage of the clostridial delivery system is that not only is it tumor specific, it is also safe in humans. As cited by Liu et al.,115 Mose et al. injected themselves with spores of C. sporogenes (a strain later named C. oncolyticum) and experienced a mild fever as the only side effect. The safety of C. oncolyticum was substantiated in clinical trials with cancer patients who had a variety of solid tumors119 and also in more recent trials in noncancer patients with a C. beijerinckii strain.120 An additional advantage is that injection of spores did not appear to elicit an immune response.115 Another example of hypoxia-selective GDEPT is prodrug 70 (CB 1954, Scheme 29). CB 1954 was originally synthesized over 30 years ago. It exhibits dramatic and

N

N NO2 H2N

NHOH

Reductase

Thioester (e.g, acetyl CoA)

H2N O

NO2

70 5-(Aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954)

O

NO2 70a O

N N

O

H2N O

NO2

70b (DNA cross-linking agent)

Scheme 29. Bioactivation of CB 1954 by reductases.

highly specific antitumor activity against rat Walker 256 carcinoma cells.121 The antitumor effect is due to efficient drug activation by rat DT-diaphorase.122 CB 1954 entered clinical trials in 1970s, but little antitumor activity was observed,

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METABOLIC ACTIVATION AND DRUG TARGETING

as human DT-diaphorase is much less active in the reduction of CB 1954 than the rat enzyme. Recent studies revealed that an amino acid difference at residue 104 between the human and rat enzymes is responsible for the catalytic difference to CB 1954.123 A nitroreductase (NTR) gene isolated from E. coli has been demonstrated to activate the prodrug CB 1954 to its toxic form approximately 90-fold more rapidly than rat DT-diaphorase, suggesting the possibility of using CB 1954 with NTR in ADEPT124,125 and GDEPT.126–130 Shibata et al. successfully transfected human HT 1080 tumor cells with the E. coli NTR gene.131 The transfected human tumor cells expressed E. coli NTR in a time- and concentration-dependent manner under a hypoxic condition while expressing only trace levels under an aerobic condition, indicating that the enzyme’s expression is induced by hypoxia. No NTR was observed with wild-type HT1080 cells. The expression of NTR conferred increased sensitivity of human tumor cells to CB 1954 both in vitro and in vivo. The IC50 value obtained with the transfected cells was reduced by 40- to 50-fold compared to the IC50 value with the wild cells in an in vitro experiment under a hypoxic condition. Significantly, no sensitivity difference was observed between the transfected and wild HT 1080 cells under an aerobic condition, consistent with the notion that NTR is induced under hypoxic conditions. A significant tumor growth delay was also observed in mice implanted with transfected clones of HT 1080 cells under a hypoxic condition. Similar to the in vitro result, no sensitivity difference to CB 1954 was observed when the in vivo experiment was conducted under an aerobic condition. Other examples of GDEPT include using CPG2 to activate nitrogen mustard,99,132 DT-diaphorase, and NADPH-cytochrome c (P450) reductase to activate mitomycin,133 CYP2B1 to activate cyclophosphamide and ifosfamide,94 and pyrimidine nucleoside phosphorylase (PyNPase) to activate 50 -DFUR to 5-FU.134

11.4. SUMMARY The search for tumor-selective prodrugs has been a long and ongoing effort. Numerous approaches have been developed based on the exploitation of the biochemical differences between cancer and normal cells. The differences can be further amplified by the use of ADEPT and GDEPT. Among the various approaches used, anticancer prodrugs based on tumor hypoxia and metastasis appear to be the most extensively explored, and some of these prodrugs have proceeded to clinical trials. ADEPT and GDEPT provide the advantage of increased tumor selectivity. The high immune response and cost are the major disadvantage of these two approaches. However, the advances in genetic technology have prompted very active research in GDEPT which is expected to play a more significant role in tumor-selective anticancer prodrug development. Overall, tumor-selective anticancer prodrugs have been shown to be an effective way to improve therapeutic efficacy and reduce systemic side effects.

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12 ULTRASOUND-MEDIATED DRUG DELIVERY KA-YUN NG* Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, CO 80262

TERRY O. MATSUNAGA Imarx Therapeutics Inc., Tucson, AZ 85719

12.1. Introduction 12.2. Background 12.2.1. Basic physics of sound 12.2.2. History of the development of ultrasound in medicine 12.2.3. Therapeutic applications of ultrasound in drug delivery 12.3. Ultrasound applications in drug delivery 12.3.1. Chemotherapeutic drugs 12.3.1.1. Ultrasound in cancer therapy 12.3.1.2. Mechanisms of ultrasound-enhanced chemotoxicity 12.3.1.3. Ultrasound as adjuvant to overcome the clinical effects of multidrug resistance 12.3.2. Thrombolytic drugs 12.3.2.1. Thrombosis and its clinical implications 12.3.2.2. Limitations of present treatment modalities for thrombosis 12.3.2.3. Ultrasound and its role in the treatment of thrombosis 12.3.2.4. Mechanisms of ultrasound-enhanced thrombolysis 12.3.2.5. Lower-frequency ultrasound in ultrasound-enhanced thrombolysis *

To whom correspondence and reprint requests should be addressed at the Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Campus Box C-238, 4200 East Ninth Avenue, Denver, CO 80262. Phone: 303-315-6997; Fax: 303-315-0274; e-mail: [email protected].

Drug Delivery: Principles and Applications Edited by Binghe Wang, Teruna Siahaan, and Richard Soltero ISBN 0-471-47489-4 # 2005 John Wiley & Sons, Inc. 245

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12.3.3. Proteins 12.3.3.1. Limitations of transdermal drug delivery 12.3.3.2. Ultrasound-enhanced transdermal delivery of proteins 12.3.3.3. Mechanism of enhancement of transdermal drug delivery by low-frequency ultrasound 12.3.4. Gene-based drugs 12.3.4.1. Historical perspective of gene therapy 12.3.4.2. Ultrasound-mediated gene delivery and its mechanism 12.4. Ultrasound contrast agents 12.4.1. Ultrasound contrast agents as imaging agents 12.4.2. Targeted ultrasound contrast agents 12.4.3. Ultrasound contrast agents and their roles in thrombolysis 12.4.4. Ultrasound contrast agents and their roles in gene therapy 12.5. Conclusion References

12.1. INTRODUCTION Ultrasound is best known for its imaging capability in diagnostic medicine. However, there have been considerable efforts recently to develop therapeutic uses for it. The purpose of this review is to summarize some of the recent advances made in the area of therapeutic ultrasound as they relate to drug delivery. The review will be divided into three sections. In the first section, we will briefly discuss the physical principles of ultrasound, as well as provide an overview of the history of the development of ultrasound in medicine. This will be followed by an in-depth analysis of ultrasound applications in drug delivery, with special emphases placed on the mechanistic effects of ultrasound in enhancing the delivery and efficacy of the following therapeutic drug classes: chemotherapeutic, thrombolytic, protein-, and gene-based drugs. Finally, because recent experimental evidence suggests that ultrasound contrast agents can be used as exogenous cavitation nuclei for enhancement of drug and gene delivery, we will conclude our review with an in-depth examination of this new class of agents and their roles in drug delivery. In selecting studies for this review, only scientific journals that are published in English were used. As such, some of the interesting and important ultrasound work performed in Germany, Japan, and Russia were not included. We have also emphasized findings from experiments in which the acoustical parameters are in ranges of medical ultrasound as defined in NCRP Report No. 74.1 These refer to frequencies in the megahertz (MHz) range (0.5 to 10 MHz) and average intensity between that of diagnostic and surgical applications of ultrasound. However, deviation from this norm may occur from time to time in the review in order to illustrate some recent and exciting findings on the use of low-frequency ultrasound (in the range of

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20 kHz) for drug delivery purposes. It should also be noted that this review is not written with a specific route of drug administration in mind. Rather, many drug administration routes are selected for discussion in order to demonstrate the broad application of ultrasound for drug delivery purposes.

12.2. BACKGROUND 12.2.1. Basic Physics of Sound Sound is our experience of the propagation of pressure waves through some physical elastic medium, such as air or liquid. The pressure waves are generated from mechanical vibrations, which become vibrating pressure waves, transferring energy to the medium and to the objects that the wave contacts. Because of this transfer of energy, the intensity of the sound energy is progressively decreased during its propagation. The transfer and absorption of sound energy by the objects in the path of sound propagation also causes a temperature increase. As can be seen later, this rise in temperature during medical application of ultrasound is referred to as the ‘‘thermal effect’’ of ultrasound.2 Prediction of actual temperature increase produced by ultrasound is often difficult. It requires the knowledge of not only the acoustic absorption coefficients but the conduction and convection properties of the tissues involved.2 Since discussion of these topics will be too complicated for this review, readers are referred to other technical resources for in depth review of this subject.3,4 ‘‘Ultrasound’’ refers to sound waves with frequencies above 20 kHz, which are beyond the upper limit of the human audible range.1,5–9 It is commonly classified as a nonionizing radiation along with microwaves and radiowaves. Unlike microwaves and radiowaves, which are electromagnetic radiations, ultrasound is completely mechanical in nature. However, like a beam of electromagnetic radiation, ultrasound can be reflected, refracted, diffracted, and focused. Ultrasound is generated by a transducer, which converts electrical energy to acoustical energy or vice versa. The phenomenon of conversion of mechanical acoustical energy to electricity or vice versa is commonly referred to as ‘‘piezoelectricity.’’10 In this regard, nearly all transducers use piezoelectric materials.11 These materials can be either natural crystalline solid, such as quartz, or manufactured piezoceramics, such as barium titanate and lead zirconate titanate. To produce ultrasound, a suitable voltage is applied to the transducer. When the frequency of the input voltage reaches the resonance frequency for thickness vibrations of the piezoelectric material, the piezoelectric material responds by undergoing vibrations. The vibrations are then transmitted to the environment as ultrasonic pressure waves. As noted, this phenomenon is entirely reversible. Thus, the same piezoelectric material can be used as both a transmitter and a receiver of ultrasonic waves. This property forms the basis of future ultrasound applications such as SONAR (sound navigation and ranging) and other diagnostic techniques.10

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12.2.2. History of the Development of Ultrasound in Medicine Since ultrasound in general follows the principles delineated in acoustics, its development naturally reflects the developments in acoustics. For the purpose of this review, only some of the major developments in acoustics and ultrasonics will be highlighted. For in-depth review of this area, readers are referred to a website developed by Dr. Joseph Woo,12 which contains some of the best information on the history of the study of acoustics and biomedical application of ultrasound. Galileo Galilei (1564–1642) is often said to have started the modern studies of acoustics. But several ancient philosophers played important role in laying the groundwork for future study of acoustics. As early as the sixth century BC, Pythagoras had studied the properties of vibrating strings. The study was so popular that it led to the development of a tuning system that bears his name. Two centuries later, Aristotle correctly assumed that a sound wave resonates in air through motion of the air. Soon after that, a Roman architect named Vitruvius (first century BC) correctly determined the mechanism for the movement of sound waves that contributed significantly to the acoustic design of theatres. And in the sixth century AD, Boethius, a Roman philosopher, suggested that the human perception of pitch is related to the physical property of frequency. Galilei further elevated the study of vibrations by correlating the relationship between pitch and frequency using strict scientific standards. Then in 1877, the field of acoustics was catapulted to new heights with the publication of the famous treatise ‘‘The Theory of Sound’’ in England by Lord Rayleigh, in which he delineated the fundamental physics of sound vibrations (waves), transmission, and reflections. Perhaps the most important breakthrough in ultrasound technique came in 1880 when the piezoelectric effects in certain crystals were discovered by the French scientist Pierre Curie and his brother Jacques Curie.10 They observed that an electric potential was produced when mechanical pressure was exerted on a quartz crystal such as Rochelle salt; conversely, the application of an electric charge produced deformation in the crystal, causing it to vibrate. This work forms the basis of echosounding technique and lays the foundation for future ultrasound applications such as SONAR and other diagnostic techniques. Basic to this technique is a ‘‘pulseecho’’ operation in which short pulses of ultrasound are emitted from one or more transmitting transducers. Upon impinging on an object, pulses are reflected as echo waves and detected by receiving transducers. The first large-scale application of SONAR was in antisubmarine warfare. In the late 1940s and mid-1950s, pulse-echo methods (similar to those used in SONAR) were introduced and applied to diagnostic medicine. This effort came, in part, from the desire of physicians to reduce dependence on other ionizing methods, which were viewed as being unsafe for imaging purposes, such as X-ray. Interestingly, applications of ultrasound as a method of therapy predated diagnostic ultrasound, utilizing its heating and disruptive effects on animal tissues. The destructive ability of high-intensity ultrasound had been recognized in the 1920s from the time of Langevin (an eminent French physicist) when he noted destruction of schools of fishes in the sea and pain induced in the hand when placed

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TABLE 12.1 Applications

Exposure Intensity Levels in Medical

Category Surgical Therapeutic Diagnostic

Typical Intensity Range >10 W/cm2 0.5–3 W/cm2 0.0001–0.5 W/cm2

Source: Reproduced from Ref. 1.

in a water tank insonated with high-intensity ultrasound. In the 1930s and 1940s, ultrasound was introduced as a method of therapy in Europe, wherein irradiation with ultrasound was used to generate heat. Thus, tissue deep in the body can be warmed by this means. In the 1940s, ultrasound had slowly evolved to become a neurosurgical tool, being used to destroy part of the basal ganglia in patients with parkinsonism. Ultrasonics was also extensively used in physical and rehabilitation therapy. The earliest reported use of ultrasound as a physical therapy in the United States was at the University of Colorado in 1953 by Jerome Gersten for treatment of patients with rheumatoid arthritis. For the past 50 years or so, ultrasound has been accepted as a useful tool in physical therapy. It should be noted that, while the intensity of ultrasound used for therapeutic (such as physical therapy) and diagnostic purposes is considered noninvasive, the intensity levels needed for such applications differ.1 In the case of diagnostic ultrasound, intensities in the range of 0.0001 to 0.5 watts (W)/cm2 are usually used (see Table 12.1). This is substantially less than the intensity needed for therapeutic (0.5 to 3 W/cm2) or surgically based ultrasound (>10 W/cm2) applications. 12.2.3. Therapeutic Applications of Ultrasound in Drug Delivery The use of ultrasound to promote drug delivery was first reported by Fellinger and Schmid,13 who developed a successful treatment for polyarthritis by using ultrasound to drive hydrocortisone ointment into the inflamed tissues. The technique of driving molecules of drug across the percutaneous barrier to the target area using ultrasonic perturbation is termed ‘‘sonophoresis’’ or ‘‘phonophoresis.’’ Since then, a wide variety of drug/ultrasound combinations have been implemented for sonophoresis.14 Most recently, ultrasound application has been used to promote delivery of high molecular weight proteins through intact skin.15–17 In addition to sonophoresis research, ultrasound has been shown to enhance the effects of several therapeutic drug classes, including chemotherapeutic, thrombolytic, and gene-based drugs. Furthermore, ultrasound contrast agents, which were originally developed for diagnostic ultrasound, have been shown to augment the delivery and effectiveness of certain drugs. These ultrasound contrast agents can also be used as drug carriers for responsive and targeted drug delivery in the presence of ultrasound insonation.18,19 In the following sections, we will offer an in-depth analysis of ultrasound

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applications in drug delivery of chemotherapeutic, thrombolytic, protein-, and gene-based drugs, and some of the recent applications of ultrasound contrast agents in drug delivery.

12.3. ULTRASOUND APPLICATIONS IN DRUG DELIVERY 12.3.1. Chemotherapeutic Drugs 12.3.1.1. Ultrasound in Cancer Therapy The use of ultrasound to enhance cancer therapy has been the subject of numerous biological and clinical investigations. In most of these studies, ultrasound has been used as an agent to induce hyperthermia for either direct treatment of small and localized cancerous tumors20–27 or as adjuvant therapy to increase the efficacy of radiotherapy28–30 and chemotherapy.31– 41 In the first application, acoustic intensities of thousands of W/cm2 and temperatures in excess of 98
C are often used to coagulate cancerous tissues.20,21,42 This form of hyperthermia is often referred to as ‘‘thermal therapy’’ because of its extreme temperature and energy.43 Alternatively, lower ultrasound intensities (0.2 to several W/cm2) produce a mild increase in temperature (41 to 45
C), and enhance the cytotoxicity of radiation therapy28–30 and chemotherapy.31,33,36,40,41 The enhancing effect of ultrasound on radiation therapy has been linked to the radiosensitization effect of hyperthermia, which increases radiation damage and prevents subsequent repair.28 However, the precise mechanism for ultrasoundenhanced chemotoxicity is still the subject of debate. 12.3.1.2. Mechanisms of Ultrasound-Enhanced Chemotoxicity The mechanisms proposed to account for ultrasound-enhanced chemotoxicity include (1) (nonthermal) and (2) thermal mechanisms. The nonthermal effects of ultrasound may refer to conditions wherein ultrasound enhances chemotoxicity at or below normal physiological temperatures.32–35,37–39 In experiments carried out at above physiological temperatures (i.e., >37
C), it can also refer to chemotoxicity enhancement that cannot be accounted for by the effects of heat alone (see Table 12.2).31,33,36,40,41 The nonthermal effects of ultrasound on a biological system can occur via one or a combination of three mechanisms: (1) cavitation, (2) radiation pressure, and (3) acoustic microstreaming.1,7,44 ‘‘Cavitation’’ refers to activity associated with small gaseous bubbles. Radiation pressure is time-averaged pressure elevations at any point in a medium traversed by ultrasound. They can act on and cause translational displacement of any discrete body by a steady force referred to as ‘‘radiation force.’’ When radiation force acts on the elements of a homogeneous medium, such as a fluid, it causes the formation of a steady circulatory flow or acoustic streaming. This streaming can be both large and small in scale. In small-scale acoustic streaming (microstreaming), micro-scale eddying (on the order of a micron) in the vicinity of a small vibrating object occurs. Microstreaming is readily set up near gas bubbles; thus it is also closely related to cavitation. The

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TABLE 12.2

Effect of Ultrasound Exposure on the Activity of Chemotherapy

Chemotherapeutic Agents: Enhanced Activity (þ) or No Enhancement () Ultrasound Exposure Conditions Nitrogen mustard (þ) Doxorubicin (þ); daunomycin (?) Doxorubicin(þ); amphotericin B (þ); cisplatin (); etoposide (?) Doxorubicin (þ) Doxorubicin (þ); diaziquone (þ) Doxorubicin (þ) Doxorubicin (þ); diaziquone (þ); cisplatin (); mitomycin C () Ara-C (þ)

Ref. 2

2 MHz continuous wave (CW) ultrasound; 10 W/cm ; 31 temperature: 26–42.5
C 1.92 MHz CW ultrasound; 0.66 W/cm2; temperature: 32 35
C 2.025 MHz CW ultrasound; 0.5–2 W/cm2; temperature: 33 37–43
C 2.6 MHz CW ultrasound; 2.3 W/cm2; temperature: 37
C 1.765 MHz tone burst and pulsed ultrasound; 0.25 W/cm2; temperature: 37
C 1.733 MHz CW ultrasound; 0.5–2 W/cm2; temperature: 41–43
C 1.62 MHz tone burst and pulsed ultrasound; 0.5 W/cm2; temperature: 37
C

34 35

48 kHz CW ultrasound; 0.3 W/cm2; temperature: 37
C

39

36 37, 38

nonthermal effects of ultrasound have been shown to influence cell surfaces,1,7,44 and thereby might promote cellular uptake of cytotoxic drugs and enhance chemotoxicity. However, under the conditions of the chemotoxicity studies mentioned above, all three nonthermal mechanisms could occur. Thus, it is conceivable that each or a combination of these three nonthermal mechanisms of ultrasound might play an interesting and important role in chemotoxicity enhancement. Although the nonthermal effects of ultrasound can enhance the cytotoxicity of chemotherapeutic drugs, not all drugs appear to benefit from such an approach (Table 12.2). Drugs rendered more cytotoxic include amphotericin B, daunorubicin, doxorubicin, nitrogen mustards (BCNU), and diaziquone. In contrast, the activity of cisplatin and mitomycin C are not enhanced by ultrasound. Compared to the drugs made more cytotoxic by ultrasound, mitomycin C and cisplatin are considered quite hydrophilic. As such, ultrasound tends to enhance the chemotoxicity of hydrophobic drugs. However, recent study indicated that low-frequency ultrasound (kHz range) could enhance the cytotoxicity of the hydrophilic chemotherapeutic drug, cytosine arabinoside (see Table 12.2).39 This observation is consistent with recent findings indicating that low-frequency ultrasound could enhance intracellular accumulation of small molecular and high molecular weight hydrophilic drugs.45–47 This enhancing effect is believed to be caused primarily by ultrasound-induced cavitation, which is the predominant nonthermal mechanism at the low-frequency

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range of ultrasound.1,7,44 Further, the effect exhibits good correlation with acoustic energy exposure (the product of acoustic pressure and exposure time), where an increase in energy exposure causes temporal and reversible disruption of cell membrane for enhanced intracellular drug delivery.45–47 However, since ultrasoundinduced cavitation during low-frequency ultrasound treatment is known to lead to substantial cell damage, its use may be limited to in vitro or ex vivo gene transfer or other applications where the experiments are concerned only with the fate of the remaining viable cells. The foregoing alludes to the importance of a nonthermal mechanism for ultrasound-enhanced drug uptake by cells. However, data in favor of a thermal effect for enhanced cellular drug uptake have recently been obtained in our laboratory (unpublished results). Utilizing a model lipophilic drug, rhodamine 123 (R123), in a 9L glioma cell culture system, we have shown that high-frequency (1 MHz) ultrasound treatment at below physiological temperature (i.e., 27
C) did not increase cellular uptake of R123 (see Figure 12.1). However, if the temperature was allowed to increase, increases in cellular accumulation of R123 were observed. This occurred regardless of whether the hyperthermic conditions were induced by ultrasound or nonultrasound sources. These data support the notion that a thermal rather than a nonthermal mechanism may mediate the ultrasound enhancement of cellular uptake. Accordingly, drugs whose entry into cells are limited by membrane permeability might find their permeabilities and hence cytotoxicities enhanced at elevated temperatures. Indeed, an example of such behavior exists for adriamycin or doxorubicin.48 Aside from increasing membrane permeability to drugs, the ability of ultrasound to heat biological tissue also increases the cytotoxicity of certain

Fluorescence intensity/protein (µg)

50

40

30

20

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0 US41°C

HT

37°C

US27°C

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Figure 12.1. Effect of 41
C (HT), continuous wave (CW) ultrasound at 41
C (US41
C: 1 MHz at 0.4 W/cm2), CW ultrasound at 27
C (US27
C: 1 MHz at 0.4 W/cm2) on cellular uptake of R123 by 9L glioma cells in vitro. Cellular uptake of R123 was expressed as fluorescence intensity per mg of protein. For the study, ultrasound exposure was continued for 20 minutes. As controls, another groups of cells were treated at either 37
C or 27
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253

drugs, such as nitrosoureas, whose cytotoxic reactions follow an Arrhenius-type law, i.e., the rate constant governing the reaction increases more or less exponentially with temperature.48 12.3.1.3. Ultrasound as Adjuvant to Overcome the Clinical Effects of Multidrug Resistance Multidrug resistance (MDR) represents a major obstacle to successful chemotherapy of metastatic diseases.49 One of the best-understood mechanisms of MDR is the overexpression of P-glycoprotein (P-gp).50 This 170 kDa plasma membrane protein belongs to a larger family of ATP-binding cassette proteins and confers resistance to tumor cells by extruding many structurally and functionally unrelated hydrophobic anticancer drugs using the energy of ATP hydrolysis.51 High-frequency ultrasound (MHz) ranging from 0.5 W/cm2 to several watts could increase treatment temperatures and enhance the activity of several hydrophobic drugs (Table 12.1). It would appear that ultrasound-induced heat could have potential in increasing cellular uptake of P-gp substrates and thereby reducing the clinical effects of MDR. We recently conducted studies to examine this possibility by investigating how mild heat (41
C) produced by a nonultrasound heat source (HT) or 1 MHz ultrasound (ultrasound-induced mild heat (USMH)) could affect (1) the cellular uptake of P-gp substrates, R123 and doxorubicin and (2) the cytotoxicity of doxorubicin in the parent and P-gp-overexpressing variants of two human cancer cell lines, MV522 and KB.40 Our results indicated that both experimental conditions led to increased cellular uptake of R123 and doxorubicin in the parent and P-gp-overexpressing variants of the two cell lines (Figure 12.2). In parallel, the cytotoxicity of doxorubicin, was also enhanced. Importantly, the enhancement of uptake of R123 and doxorubicin, and of the cytotoxicity of doxorubicin produced by HT or USMH, was far better than that produced by the P-gp modulator, verapamil (Figure 12.3). Because heating has been shown to induce a reduction in P-gp expression, leading to inhibition of drug efflux and enhancement of intracellular drug uptake, we recently examined the expression and activity of P-gp in both heat-treated and untreated cells. Both the expression level52 and the activity of P-gp were unaffected by the heat treatment.53 These results imply that a mechanism other than modulation of P-gp activity might be responsible for the enhanced drug accumulation by USMH. Our recent data suggest that the site of action of USMH might be the cell membrane and that USMH selectively enhances the permeability of hydrophobic but not hydrophilic drugs.53 If USMH were to increase membrane permeability to hydrophobic drugs, it is possible that the enhanced drug entry rate might temporarily overwhelm P-gp and lead to more cellular drug accumulation. It is reckoned that if the enhanced drug uptake could be maintained in the P-gpexpressing cells longer, an augmented binding of the anticancer drug to its target and, therefore, increased cytotoxicity might be obtained. To that end, we tested the effects of combining USMH, which enhances cellular drug uptake, with PSC 833, which modulates P-gp activity and hence promotes cellular drug retention, on cytotoxicity of doxorubicin in the parent and MDR cells.41 The results of this study are shown in Figure 12.4. As shown in this figure, cytotoxicity of anticancer drugs

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MDR variants

Parent MV522 cells

6

* R123 DOX

4 2 0

Drug only

10 8

Drug + ver

Drug + HT

*

6 4 2 0

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Drug + ver

*

*

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*

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* 1

2

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KB-V-1 cells

3

* R123 DOX

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12

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*

*

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8

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3

*

Accumulation factor

Accumulation factor

10

R123 DOX

*

*

1

0

Drug only

Drug + ver

Drug + HT

Drug + USMH

Figure 12.2. Effects of HT (41
C) or USMH or verapamil (Ver) on cellular uptake of R123 or doxorubicin (DOX) by the parent and MDR variants of human MV522 (human metastatic lung carcinoma) and KB (human epidermoid carcinoma) cell lines. Cellular uptake data are presented as accumulation factors. The accumulation factor for control cells is defined as equal to 1. The accumulation factor for treated cells is defined as the ratio of cellular R123/DOX accumulation in the presence of HT, USMH, or Ver to cellular accumulation in the absence of HT, USMH, or Ver. All experiments were carried out in triplicate. * P < 0:05 compared to control values.

in P-gp-expressing cells can be significantly enhanced by the combined use of USMH and P-gp-modulating agent, where USMH increases uptake and the P-gp inhibitor reduces efflux of the cytcotoxic agent from the cell, resulting in significantly increased exposure and efficacy. These findings have significant implications for combined therapy using PSC 833 with cytotoxic anticancer drugs. For instance, PSC 833 has been found to alter the pharmacokinetic profile of the concomitant anticancer drugs, which leads to unexpected toxicity.54 As a result, dosage reductions of anticancer drugs are often needed to avert unexpected toxicity. In the context of the present study, this reduction in dosage may be compensated for by the USMH, which specifically increases cellular uptake of the cytotoxic agent into the tumor.

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MDR variants

Parent

MV522/Q6 cells

MV522 cells 100

*

*

80

60

% inhibition

% inhibition

80

100

40 20

DOX DOX USMH DOX DOX + USMH + HT + ver

60 40 20

DOX DOX USMH DOX DOX + USMH + HT + ver

Ver

KB-3-1 cells

100

*

Ver

KB-V-1 cells

100 80

*

60

% inhibition

% inhibition

*

0

0

80

*

40 20

60

*

*

40 20 0

0 DOX DOX USMH DOX DOX + ver + USMH + HT

Ver

DOX DOX USMH DOX DOX + ver + USMH + HT

Ver

Figure 12.3. Effects of HT (41
C) or USMH or verapamil (Ver) on cytotoxicity enhancement of doxorubicin (DOX) in the parent or MDR variants of human MV522 and KB cell lines. Data are expressed as % inhibition calculated by the following formula: % inhibition ¼ [1  (counts of viable drug-exposed cells/counts of viable non-drug-exposed cells)]  100. Cell viability was determined by hemocytometry technique after trypan blue staining. All experiments were carried out in triplicates. *P < 0:05 compared to control values.

12.3.2. Thrombolytic Drugs 12.3.2.1. Thrombosis and Its Clinical Implications As a normal hemostatic response to limit hemorrhage from microscopic or macroscopic vascular injury, the body undergoes a process termed ‘‘local thrombosis.’’ Specific proenzymes and proteins, platelets, and calcium participate in this process. The end result is the formation of insoluble fibrin, which mechanically blocks the flow of blood through ruptured vessels.55 Thrombosis is usually counterbalanced by physiological anticoagulation and thrombolysis. Under normal conditions, the thrombus is confined to the area of vessel injury and rarely obstructs flow to critical areas. However,

B

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25

50

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125

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25

50

75

100

125

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Control

+ PSC

+ PSC + PSC + DOX

+ DOX + PSC + DOX

KB-V-1, 37°C C KB-V-1, 41°C C (USHT)

+ DOX

KB-3-1, 37°C C KB-3-1, 41˚ 41°C C (USHT)

D

C

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+ PSC

+ PSC

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+ DOX

+ PSC + DOX

MV-522/Q6, 37°C MV-522/Q6, 41°C (USHT)

+ DOX

MV-522, 37°C MV-522, 41°C (USHT)

Figure 12.4. Effect of combined use of USMH (41
C) and PSC 833 on cytotoxicity of doxorubicin in the parent and MDR variant of MV522 and KB lines. Data were expressed as % inhibition calculated by the following formula: % inhibition ¼ [1  (counts of viable drug-exposed cells/counts of viable non-drug-exposed cells)]  100. All experiments were carried out in triplicate (mean  standard deviation).

% inhibition of proliferation

% inhibition of proliferation

A % inhibition of proliferation % inhibition of proliferation

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257

under pathological conditions, the thrombus can dislodge from the area of vessel injury and enters the blood flow. The thrombus can then lodge in a critical vessel, and can obliterate valves and other structures that are essential to normal hemodynamic function. Abnormal thrombosis can occur in any vessel at any location in the body. The principal clinical syndromes that result are acute myocardial infarction, deep vein thrombosis, pulmonary embolism, acute non-hemorrhagic stroke, and acute peripheral arterial occlusion.55 12.3.2.2. Limitations of Present Treatment Modalities for Thrombosis The success of treatment of abnormal thrombosis depends on how rapidly one can restore blood flow to the obstructed vessels, because tissue death is closely related to the duration of ischemia.55 Although recanalization of acutely thrombosed arteries can be achieved by mechanical interventions, such as balloon angioplasty,56 this technique is not considered feasible for conditions such as stroke. Reperfusion by such techniques have serious limitations, including the requirement for specialized facilities and highly trained personnel, and these procedures often result in more complications in older patients.57–62 Consequently, drug-induced thrombolysis has surfaced as an alternative for treatment of pathological thrombosis.63 Thrombolytic agents available today are serine proteases, such as tissue type plasminogen activator (t-PA), urokinase, and streptokinase, that work by converting plasminogen to the natural thrombolytic agent, plasmin. Plasmin lyses thrombotic vascular occlusions by degrading fibrinogen and fibrin contained in a blood clot. Thrombolytic agents, however, also have limited success in recanalizing thrombotically occluded arteries.64,65 For instance, in ischemic stroke treatment, thrombolytic therapy with t-PA demonstrated a small but significant improvement in neurological outcome in selected patients. As such, there is a need to enhance the effectiveness of thrombolytic agents by shortening the time to reperfusion. 12.3.2.3. Ultrasound and Its Role in the Treatment of Thrombosis Among the first to study the application of ultrasound to enhance the effectiveness of thrombolytic agents was Kudo,66 who showed that, when used together with recombinant tPA, ultrasound can accelerate recanalization of thrombotically occluded arteries in vivo. This observation was later confirmed by other investigators in various in vitro studies67–76 and in animal models of arterial9,77 and small-vessel thrombosis.78,79 Consistent findings among these studies are: (1) the rate of thrombolytic enhancement was directly related to the temporal average intensity of the field; (2) the ultrasound-induced enhancement of thrombolysis was greater at higher thrombolytic concentrations; (3) a lower concentration of thrombolytic agent in the presence of ultrasound could induce more thrombolysis than a higher concentration in the absence of ultrasound; (4) enhancement was due primarily to accelerated enzymatic (thrombolytic) action rather than mechanical disruption of the clot;80 and (5) enhancement was not limited to t-PA-specific drugs; the activity of other thrombolytic drugs, such as urokinase and streptokinase, was also increased.80

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12.3.2.4. Mechanisms of Ultrasound-Enhanced Thrombolysis Ultrasound is known to have several biological effects, depending on the emission characteristics. At higher energy levels, ultrasound alone has a thrombolytic effect. This effect is already used for clinical purposes in interventional therapy using ultrasound catheters. At high intensities, ultrasound delivered via a transducer causes mechanical fragmentation through direct mechanical contact. In contrast, the use of lowerintensity ultrasound to accelerate enzymatic thrombolysis without mechanical disruption of the thrombus offers a conceptually different approach to thrombolytic therapy. The mechanisms by which ultrasound enhances enzymatic thrombolysis are multiple and relate primarily to transport (refer to Francis80 for a recent review). Using 1 MHz ultrasound at 4 W/cm2, Francis et al.81 demonstrated that ultrasound significantly increases the rate of uptake and penetration of t-PA into the clots. Ultrasound also increases thrombolysis by pressure-mediated perfusion of thrombolytic agents into the matrix. For instance, Siddiqi et al.82 examined the effects of ultrasound on fluid permeation through purified fibrin gels and found that at 2 W/cm2, 1 MHz ultrasound increased flow through the gel. Flow through the gel was dependent on the ultrasound intensity (hence acoustic pressure) and the resistance of the fibrin gel (a property determined by the fibrin fiber structure). In the context of this study, ultrasound promotes thrombolysis by altering the fiber structure. Indeed, when the ultrastructure of fibrin gel after ultrasound treatment was examined using scanning electron microscopy, significant changes in structure were observed.83 Such alterations could modify flow resistance as well as create additional binding sites for thrombolytic enzymes, thereby improving thrombolytic efficacy. In order to study if inertial cavitation might be the mechanism by which ultrasound enhanced the dissolution of a blood clot when the clot was exposed to a thrombolytic agent, Everbach and Francis84 examined the dissolution of radiolabeled fibrin clots exposed to 1 MHz ultrasound in a rotating sample holder, a condition which enhances ultrasound-induced nonthermal effects such as inertial cavitation.44 To suppress acoustic cavitation, the exposure tank was positioned in a hyperbaric chamber capable of pneumatic pressurization to 10 atmospheres. In these studies, static pressure could only reduce the acceleration of thrombolysis by one-half, suggesting that other processes in addition to inertial cavitation might be responsible for the enhanced effect of ultrasound on thrombolysis. Hence, much work is needed to shed further light on the mechanism of this phenomenon. 12.3.2.5. Lower-Frequency Ultrasound in Ultrasound-Enhanced Thrombolysis The pioneering work by Kudo66 used relatively low-frequency ultrasound (200 kHz or 0.2 MHz). However, most investigators used higher frequencies (MHz range) with subcavitation intensity. Suchkova et al.85,86 and others76,87 recently reemphasized the advantage of lower-frequency and low-intensity ultrasound (40 kHz;